real time pcr total rna  (Millipore)


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    Structured Review

    Millipore real time pcr total rna
    Expression of mGluR1 mRNA (a, c, e, and g) and protein (b, d, f, and h) in forebrain of control and EAE rats at different times post-immunization (a and b) and after therapeutic treatment with antagonists of glutamate receptors: amantadine (c and d), memantine (e and f), and LY 367385 (g and h). Total <t>RNA</t> was prepared from healthy control rats, rats with EAE, and rats with EAE after therapy at the indicated d.p.i. The mGluR1 mRNA levels were determined by quantitative real-time <t>PCR</t> (see Section 2 ) and normalized against actin. Graphs (a), (c), (e), and (g) present the results expressed as percentage of control from four independent experiments. * P
    Real Time Pcr Total Rna, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 132 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 92 stars, based on 132 article reviews
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    real time pcr total rna - by Bioz Stars, 2020-08
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    Images

    1) Product Images from "Modulation of Neurological Deficits and Expression of Glutamate Receptors during Experimental Autoimmune Encephalomyelitis after Treatment with Selected Antagonists of Glutamate Receptors"

    Article Title: Modulation of Neurological Deficits and Expression of Glutamate Receptors during Experimental Autoimmune Encephalomyelitis after Treatment with Selected Antagonists of Glutamate Receptors

    Journal: BioMed Research International

    doi: 10.1155/2013/186068

    Expression of mGluR1 mRNA (a, c, e, and g) and protein (b, d, f, and h) in forebrain of control and EAE rats at different times post-immunization (a and b) and after therapeutic treatment with antagonists of glutamate receptors: amantadine (c and d), memantine (e and f), and LY 367385 (g and h). Total RNA was prepared from healthy control rats, rats with EAE, and rats with EAE after therapy at the indicated d.p.i. The mGluR1 mRNA levels were determined by quantitative real-time PCR (see Section 2 ) and normalized against actin. Graphs (a), (c), (e), and (g) present the results expressed as percentage of control from four independent experiments. * P
    Figure Legend Snippet: Expression of mGluR1 mRNA (a, c, e, and g) and protein (b, d, f, and h) in forebrain of control and EAE rats at different times post-immunization (a and b) and after therapeutic treatment with antagonists of glutamate receptors: amantadine (c and d), memantine (e and f), and LY 367385 (g and h). Total RNA was prepared from healthy control rats, rats with EAE, and rats with EAE after therapy at the indicated d.p.i. The mGluR1 mRNA levels were determined by quantitative real-time PCR (see Section 2 ) and normalized against actin. Graphs (a), (c), (e), and (g) present the results expressed as percentage of control from four independent experiments. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Expression of mGluR5 mRNA (a, c, e, and g) and protein (b, d, f, and h) in forebrain of control rats and rats with EAE at different times post-immunization (a and b) and after therapeutic treatment with antagonists of glutamate receptors: amantadine (c and d), memantine (e and f), and MPEP (g and h). Total RNA was prepared from healthy control rats, rats with EAE, and rats with EAE after therapy at the indicated d.p.i. The mGluR5 mRNA levels were determined by quantitative real-time PCR (see Section 2 ) and normalized to actin. Graphs (a), (c), (e), and (g) present the results expressed as percentage of control from four independent experiments. * P
    Figure Legend Snippet: Expression of mGluR5 mRNA (a, c, e, and g) and protein (b, d, f, and h) in forebrain of control rats and rats with EAE at different times post-immunization (a and b) and after therapeutic treatment with antagonists of glutamate receptors: amantadine (c and d), memantine (e and f), and MPEP (g and h). Total RNA was prepared from healthy control rats, rats with EAE, and rats with EAE after therapy at the indicated d.p.i. The mGluR5 mRNA levels were determined by quantitative real-time PCR (see Section 2 ) and normalized to actin. Graphs (a), (c), (e), and (g) present the results expressed as percentage of control from four independent experiments. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Expression of mRNA of NMDARs (a, c, and e) and protein (b, d, and f) in forebrain of control and EAE rats at different times post-immunization (a and b) and after therapeutic treatment with antagonists of NMDA receptors: amantadine (c and d) and memantine (e and f). Total RNA was prepared from healthy control rats, rats with EAE, and rats with EAE after therapy at the indicated d.p.i. Levels of NMDA mRNAs were determined by quantitative real-time PCR (see Section 2 ) and normalized to actin. Graphs (a), (c), and (e) present the results expressed as percentage of control from four independent experiments. * P
    Figure Legend Snippet: Expression of mRNA of NMDARs (a, c, and e) and protein (b, d, and f) in forebrain of control and EAE rats at different times post-immunization (a and b) and after therapeutic treatment with antagonists of NMDA receptors: amantadine (c and d) and memantine (e and f). Total RNA was prepared from healthy control rats, rats with EAE, and rats with EAE after therapy at the indicated d.p.i. Levels of NMDA mRNAs were determined by quantitative real-time PCR (see Section 2 ) and normalized to actin. Graphs (a), (c), and (e) present the results expressed as percentage of control from four independent experiments. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    2) Product Images from "Synergism between cAMP and PPAR γ Signalling in the Initiation of UCP1 Gene Expression in HIB1B Brown Adipocytes"

    Article Title: Synergism between cAMP and PPAR γ Signalling in the Initiation of UCP1 Gene Expression in HIB1B Brown Adipocytes

    Journal: PPAR Research

    doi: 10.1155/2013/476049

    The effect of forskolin and rosiglitazone on mRNA expression of (a) UCP1, (b) PGC1 α , (c) Cidea, (d) PRDM16, (e) C/EBP β , and (f) PPAR γ is mediated by PKA and PPAR γ dependent pathways. HIB-1B cells were grown to confluence and then treated with H89 (10 μ M) for 1 hour or GW9662 (30 μ M) for 3 hours prior to and during addition of rosiglitazone (Rosi) (10 μ M) for 24 hours, or forskolin (Fosk) (10 μ M) for the final 3 hours of rosiglitazone treatment, before RNA extraction, as indicated. All drugs were added in serum-free medium. Controls were treated with DMSO. Gene expression levels were analysed by quantitative real-time PCR and normalized against 36B4 expression. Error bar means the mean ± SEM of triplicate observations within a single experiment performed in triplicate. *** Significant difference P
    Figure Legend Snippet: The effect of forskolin and rosiglitazone on mRNA expression of (a) UCP1, (b) PGC1 α , (c) Cidea, (d) PRDM16, (e) C/EBP β , and (f) PPAR γ is mediated by PKA and PPAR γ dependent pathways. HIB-1B cells were grown to confluence and then treated with H89 (10 μ M) for 1 hour or GW9662 (30 μ M) for 3 hours prior to and during addition of rosiglitazone (Rosi) (10 μ M) for 24 hours, or forskolin (Fosk) (10 μ M) for the final 3 hours of rosiglitazone treatment, before RNA extraction, as indicated. All drugs were added in serum-free medium. Controls were treated with DMSO. Gene expression levels were analysed by quantitative real-time PCR and normalized against 36B4 expression. Error bar means the mean ± SEM of triplicate observations within a single experiment performed in triplicate. *** Significant difference P

    Techniques Used: Expressing, RNA Extraction, Real-time Polymerase Chain Reaction

    3) Product Images from "Aberrant KDM5B expression promotes aggressive breast cancer through MALAT1 overexpression and downregulation of hsa-miR-448"

    Article Title: Aberrant KDM5B expression promotes aggressive breast cancer through MALAT1 overexpression and downregulation of hsa-miR-448

    Journal: BMC Cancer

    doi: 10.1186/s12885-016-2108-5

    KDM5B is a functional target of hsa-miR-448 in TNBC cells. a KDM5B, MALAT1 and hsa-miR-448 protein or nucleotide input sequence obtained from the National Center for Biotechnology Information (NCBI) database, http://www.ncbi.nlm.nih.gov/ . b Predicted KDM5B 3′-UTR binding site for hsa-miR-448. The hsa-miR-448 seed region alignment with KDM5B 3′-UTR is shown. c Upper panel: Sequence-based prediction of KDM5B-hsa-miR-448 interaction, with random forest, RF classifier score of 0.7 and support vehicle machine, SVM classifier score of 0.81. Lower panel: Sequence-based predicted interaction of KDM5B-MALAT1-hsa-miR-448, with RF classifier score of 0.75 and SVM classifier score of 0.997. * Interaction probabilities generated by RPISeq range from 0 to 1. In performance evaluation experiments, predictions with probabilities > 0.5 were considered “positive,” i.e., indicating that the corresponding RNA and protein are likely to interact. d The effect of KDM5B knockdown on the expression of KDM5B transcripts using qRT-PCR. e Loss of KDM5B function altered hsa-miR-448 mRNA expression in MDA-MB-231 cells. MDA-MB-231 cells were infected with scramble (231 V), shKDM5B clone II (231 II) or clone III (231 III) for 72 h. The transcript levels of KDM5B and hsa-miR-448 were assessed by qRT-PCR. f The mRNA expression of KDM5B and hsa-miR-448 is inversely correlated. Data are representative of 3 independent experiments and analyzed by student’s t-test. All data are shown as mean ± SEM. ** p
    Figure Legend Snippet: KDM5B is a functional target of hsa-miR-448 in TNBC cells. a KDM5B, MALAT1 and hsa-miR-448 protein or nucleotide input sequence obtained from the National Center for Biotechnology Information (NCBI) database, http://www.ncbi.nlm.nih.gov/ . b Predicted KDM5B 3′-UTR binding site for hsa-miR-448. The hsa-miR-448 seed region alignment with KDM5B 3′-UTR is shown. c Upper panel: Sequence-based prediction of KDM5B-hsa-miR-448 interaction, with random forest, RF classifier score of 0.7 and support vehicle machine, SVM classifier score of 0.81. Lower panel: Sequence-based predicted interaction of KDM5B-MALAT1-hsa-miR-448, with RF classifier score of 0.75 and SVM classifier score of 0.997. * Interaction probabilities generated by RPISeq range from 0 to 1. In performance evaluation experiments, predictions with probabilities > 0.5 were considered “positive,” i.e., indicating that the corresponding RNA and protein are likely to interact. d The effect of KDM5B knockdown on the expression of KDM5B transcripts using qRT-PCR. e Loss of KDM5B function altered hsa-miR-448 mRNA expression in MDA-MB-231 cells. MDA-MB-231 cells were infected with scramble (231 V), shKDM5B clone II (231 II) or clone III (231 III) for 72 h. The transcript levels of KDM5B and hsa-miR-448 were assessed by qRT-PCR. f The mRNA expression of KDM5B and hsa-miR-448 is inversely correlated. Data are representative of 3 independent experiments and analyzed by student’s t-test. All data are shown as mean ± SEM. ** p

    Techniques Used: Functional Assay, Sequencing, Binding Assay, Generated, Expressing, Quantitative RT-PCR, Multiple Displacement Amplification, Infection

    4) Product Images from "Interplay between PTB and miR-1285 at the p53 3′UTR modulates the levels of p53 and its isoform Δ40p53α"

    Article Title: Interplay between PTB and miR-1285 at the p53 3′UTR modulates the levels of p53 and its isoform Δ40p53α

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx630

    PTB binds specifically to p53 3′UTR. ( A ) α- 32 P-labeled 3′UTR (Lanes 3,4) and α- 32 P-labeled 5′UTR (lanes 5 and 6) were UV cross-linked with increasing concentration of PTB (250 ng, 500 ng), lane 2 is no protein (NP) and lane 1 represents the MW marker in kDa. ( B ) Competition UV crosslinking of PTB and α- 32 P-labeled 3′UTR with 100- and 200-fold molar excess of either unlabeled p53 3′UTR RNA (lanes 4 and 5), unlabeled p53 5′UTR (lanes 6 and 7) or non-specific RNA (Nsp RNA) (lanes 8 and 9) showing the specificity of PTB and 3′UTR binding. Lane 1 represents the MW marker in kDa, lane 2 is no protein (NP) and lane 3 represents binding in the absence of competitor RNA (NC). ( C ) After UV cross-linking of α- 32 P-labeled p53 3′UTR RNA with A549 cell S10 extract, immunoprecipitation was carried out using the respective antibodies, followed by incubation with protein G-Sepharose beads. The beads were either used after saturation with IgG isotype control antibody (lane 2) or anti-PTB antibody (lane 3). Lane 1, no protein (NP); lane 4, 25% of the UV-cross-linked S10 extract (input). Lane 5 is UV-crosslinked recombinant PTB (500 ng) protein. Numbers to the left represent relative mobilities of the molecular mass markers. ( D ) H1299 cells were transfected with Luc-p53 3′UTR construct. After 48 h, the cells were lysed with lysis buffer. RNP complexes were immunoprecipitated with anti- PTB antibody. p53 3′UTR was detected by RT-PCR analysis ( n = 3).
    Figure Legend Snippet: PTB binds specifically to p53 3′UTR. ( A ) α- 32 P-labeled 3′UTR (Lanes 3,4) and α- 32 P-labeled 5′UTR (lanes 5 and 6) were UV cross-linked with increasing concentration of PTB (250 ng, 500 ng), lane 2 is no protein (NP) and lane 1 represents the MW marker in kDa. ( B ) Competition UV crosslinking of PTB and α- 32 P-labeled 3′UTR with 100- and 200-fold molar excess of either unlabeled p53 3′UTR RNA (lanes 4 and 5), unlabeled p53 5′UTR (lanes 6 and 7) or non-specific RNA (Nsp RNA) (lanes 8 and 9) showing the specificity of PTB and 3′UTR binding. Lane 1 represents the MW marker in kDa, lane 2 is no protein (NP) and lane 3 represents binding in the absence of competitor RNA (NC). ( C ) After UV cross-linking of α- 32 P-labeled p53 3′UTR RNA with A549 cell S10 extract, immunoprecipitation was carried out using the respective antibodies, followed by incubation with protein G-Sepharose beads. The beads were either used after saturation with IgG isotype control antibody (lane 2) or anti-PTB antibody (lane 3). Lane 1, no protein (NP); lane 4, 25% of the UV-cross-linked S10 extract (input). Lane 5 is UV-crosslinked recombinant PTB (500 ng) protein. Numbers to the left represent relative mobilities of the molecular mass markers. ( D ) H1299 cells were transfected with Luc-p53 3′UTR construct. After 48 h, the cells were lysed with lysis buffer. RNP complexes were immunoprecipitated with anti- PTB antibody. p53 3′UTR was detected by RT-PCR analysis ( n = 3).

    Techniques Used: Labeling, Concentration Assay, Marker, Binding Assay, Immunoprecipitation, Incubation, Recombinant, Transfection, Construct, Lysis, Reverse Transcription Polymerase Chain Reaction

    Interplay between the ITAFs and miRNAs at 3′UTR. ( A ) H1299 cells were co-transfected with Luc-3′UTR and either si PTB/non specific siRNA (50 nM). After 48 h, the cells were lysed with lysis buffer. RNP complexes were immunoprecipitated with anti- Ago-2/IgG antibody. p53 3′UTR was detected by RT-PCR analysis. The values for RNA immunoprecipitated with antiAgo-2 antibody in different conditions (si Nsp and si PTB) were first normalized with its respective values for RNA immunoprecipitated with IgG control antibody and then the fold change was calculated using the value for 3′UTR pulled down with anti-Ago-2 upon si Nsp transfection as the basal value ( n = 4). ( B ) H1299 cells were transfected individually with the reporter constructs Fluc (labeled as Luc), Fluc-3′UTR (labeled as Luc3′UTR). These cells were treated with doxorubicin (2 μM) for 16 h. After 24 h, the cells were harvested and checked for luciferase activity ( n = 5). ( C ) H1299 cells were transfected with plasmid expressing luciferase-p53 3′UTR (Luc 3′UTR). The cells were treated with doxorubicin (2μM) for 16 hours. After 24 hours the cells were harvested. RNP complexes were immunoprecipitated with anti-Ago-2 antibody. p53 3′UTR was detected by RT-PCR analysis. The values for RNA immunoprecipitated with anti Ago-2 antibody in different conditions (+/– DOX) were first normalized with its respective values for RNA immunoprecipitated with IgG control antibody and then the fold change was calculated using the value for 3′UTR pulled down with anti-Ago-2, without doxorubicin treatment as the basal value ( n = 5). ( D ) RNP complexes were immunoprecipitated with anti- Ago-2 antibody and endogenous p53 3′UTR was detected by RT-PCR analysis from A549 cells in presence and absence of doxorubicin (2 μM) for 16 h. The values for RNA immunoprecipitated with anti Ago-2 antibody in different conditions (+/– DOX) were first normalized with its respective values for RNA immunoprecipitated with IgG control antibody and then the fold change was calculated as in Figure 4C ( n = 5). ( E ) H1299 cells were transfected with plasmid expressing luciferase-p53 3′UTR (Luc 3′UTR) or luciferase (Fluc) alone. The cells were treated with doxorubicin (2 μM) for 16 h. After 24 h, the cells were harvested. RNP complexes were immunoprecipitated with anti-PTB antibody. Luciferase mRNA was detected by RT-PCR analysis. The values for RNA immunoprecipitated with anti PTB antibody under different conditions (+/– DOX) were first normalized with its respective values for RNA immunoprecipitated with IgG control antibody, and then the fold change was calculated using the value for 3′UTR pulled down with anti-PTB, without doxorubicin treatment as the basal value ( n = 3). ( F ) RNP complexes were immunoprecipitated with anti-PTB antibody and endogenous p53 3′UTR was detected by RT-PCR analysis from A549 cells in the presence and absence of doxorubicin (2 μM) for 16 h. The values for RNA immunoprecipitated with anti PTB antibody in different conditions (+/– DOX) were first normalized with its respective values for RNA immunoprecipitated with IgG control antibody and then the fold change was calculated as in D ( n = 4).
    Figure Legend Snippet: Interplay between the ITAFs and miRNAs at 3′UTR. ( A ) H1299 cells were co-transfected with Luc-3′UTR and either si PTB/non specific siRNA (50 nM). After 48 h, the cells were lysed with lysis buffer. RNP complexes were immunoprecipitated with anti- Ago-2/IgG antibody. p53 3′UTR was detected by RT-PCR analysis. The values for RNA immunoprecipitated with antiAgo-2 antibody in different conditions (si Nsp and si PTB) were first normalized with its respective values for RNA immunoprecipitated with IgG control antibody and then the fold change was calculated using the value for 3′UTR pulled down with anti-Ago-2 upon si Nsp transfection as the basal value ( n = 4). ( B ) H1299 cells were transfected individually with the reporter constructs Fluc (labeled as Luc), Fluc-3′UTR (labeled as Luc3′UTR). These cells were treated with doxorubicin (2 μM) for 16 h. After 24 h, the cells were harvested and checked for luciferase activity ( n = 5). ( C ) H1299 cells were transfected with plasmid expressing luciferase-p53 3′UTR (Luc 3′UTR). The cells were treated with doxorubicin (2μM) for 16 hours. After 24 hours the cells were harvested. RNP complexes were immunoprecipitated with anti-Ago-2 antibody. p53 3′UTR was detected by RT-PCR analysis. The values for RNA immunoprecipitated with anti Ago-2 antibody in different conditions (+/– DOX) were first normalized with its respective values for RNA immunoprecipitated with IgG control antibody and then the fold change was calculated using the value for 3′UTR pulled down with anti-Ago-2, without doxorubicin treatment as the basal value ( n = 5). ( D ) RNP complexes were immunoprecipitated with anti- Ago-2 antibody and endogenous p53 3′UTR was detected by RT-PCR analysis from A549 cells in presence and absence of doxorubicin (2 μM) for 16 h. The values for RNA immunoprecipitated with anti Ago-2 antibody in different conditions (+/– DOX) were first normalized with its respective values for RNA immunoprecipitated with IgG control antibody and then the fold change was calculated as in Figure 4C ( n = 5). ( E ) H1299 cells were transfected with plasmid expressing luciferase-p53 3′UTR (Luc 3′UTR) or luciferase (Fluc) alone. The cells were treated with doxorubicin (2 μM) for 16 h. After 24 h, the cells were harvested. RNP complexes were immunoprecipitated with anti-PTB antibody. Luciferase mRNA was detected by RT-PCR analysis. The values for RNA immunoprecipitated with anti PTB antibody under different conditions (+/– DOX) were first normalized with its respective values for RNA immunoprecipitated with IgG control antibody, and then the fold change was calculated using the value for 3′UTR pulled down with anti-PTB, without doxorubicin treatment as the basal value ( n = 3). ( F ) RNP complexes were immunoprecipitated with anti-PTB antibody and endogenous p53 3′UTR was detected by RT-PCR analysis from A549 cells in the presence and absence of doxorubicin (2 μM) for 16 h. The values for RNA immunoprecipitated with anti PTB antibody in different conditions (+/– DOX) were first normalized with its respective values for RNA immunoprecipitated with IgG control antibody and then the fold change was calculated as in D ( n = 4).

    Techniques Used: Transfection, Lysis, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Construct, Labeling, Luciferase, Activity Assay, Plasmid Preparation, Expressing

    Effect of knockdown of miRNAs on the expression of p53 isoforms and miRNAs interplay with PTB. ( A ) H1299 cells were co-transfected with Luc-3′UTR and anti-miRs for miR-30d/miR-1285/miR-504/miR-181 (10 nM) and Rluc (Renilla luciferase) as transfection control. After 48 h, the cells were lysed and processed for luciferase activity. The graph represents the normalized luciferase activity, i.e. Fluc/Rluc (F/R) ( n = 3). ( B ) H1299 cells were co-transfected with p53 cDNA construct having 3′UTR (5′UTR + cDNA + 3′UTR) or no 3′UTR (5′UTR + cDNA) and anti-miR-30d/anti-miR-1285 in two concentrations (10 nM and 20 nM). After 48 h, the cells were lysed and processed for immunoblotting. ( C ) Western blot analysis of cell extracts from A549-cells transfected with anti-miRs for miR-1285/miR-30d (30 nM), probed with CM1 after 48 h. Upper panel: p53 and Δ40p53; lower panel: actin. ( D ) Quantitative PCR of p21 and 14–3-3σ mRNA levels normalized to Actin in A549 cells transfected with anti-miRs for miR-1285/ miR-30d (30 nM) for 48 h ( n = 3). ( E ) H1299 cells were co-transfected with Luc-3′UTR and anti-miRs for miR-30d/miR-1285 (20 nM). After 48 h, the cells were lysed and RNP complexes were immunoprecipitated with anti-PTB antibody. RT PCR was performed for checking the levels of 3′UTR associated with PTB. The values for RNA immunoprecipitated with anti PTB antibody in different conditions (no anti-miR, anti-miR-30d and anti-miR-1285) were first normalized with its respective values for RNA immunoprecipitated with IgG control antibody and then the fold change was calculated using the value for 3′UTR pulled down with anti-PTB, without anti-miR treatment as the basal value ( n = 5). ( F ) H1299 cells were transfected with wild type Fluc -3′UTR (labeled as WT), Fluc -3′UTR mut miR-1285 M1 (3′UTR mutated for miR-1285 one binding site, labeled as M1), Fluc -3′UTR mut miR-1285 M1+M2 (3′UTR mutated for miR-1285 both binding sites, labeled as M1 + M2) and GFP expression from a GFP plasmid vector was used as transfection efficiency control. After 24 h, the cells were lysed and processed for luciferase activity. The graph shows normalized Fluc activity of different constructs ( n = 3). ( G ) H1299 cells were transfected with either wild type Fluc -3′UTR (labeled as WT) or Fluc -3′UTR mut miR-1285 M1+M2 (3′UTR mutated for miR-1285 both binding sites, labeled as M1 + M2). After 24 h, the cells were lysed and RNP complexes were immunoprecipitated with anti-PTB antibody. RT PCR was performed for checking the levels of 3′UTR associated with PTB. The values for RNA (WT and M1 + M2) immunoprecipitated with anti PTB antibody were first normalized with its respective values for RNA immunoprecipitated with IgG control antibody, and then the fold change was calculated using the value for 3′UTR pulled down with anti-PTB from WT-3′UTR transfected cells as the basal value ( n = 6). ( H ) H1299 cells were transfected with either wild type Fluc -3′UTR (labeled as WT) or Fluc -3′UTR mut miR-1285 M1+M2 (3′UTR mutated for miR-1285 both binding sites, labeled as M1 + M2). After 24 h, the cells were lysed and RNP complexes were immunoprecipitated with anti-Ago-2 antibody. q-RT PCR was performed for checking the levels of 3′UTR associated with Ago-2. The values for RNA (WT and M1+M2) immunoprecipitated with anti Ago-2 antibody were first normalized with its respective values for RNA immunoprecipitated with IgG control antibody and then the fold change was calculated using the value for 3′UTR pulled down with anti-Ago-2 from WT-3′UTR transfected cells as the basal value ( n = 6). ( I ) Toe-printing analysis to map the contact points of PTB on p53 3′UTR. p53 3′UTR RNA was incubated in absence (lane 7) and presence (lanes 5 and 6) of increasing concentrations of purified recombinant PTB (200 ng, 400 ng). The RNA in the ribonucleoprotein complexes were reverse transcribed using 3R3 reverse primer (p53 3′UTR region 3 reverse primer) and the resulting cDNAs were resolved in 8% acrylamide–8 M urea PAGE in parallel with a sequencing reaction. The cDNA products terminated at the sites due to protein binding is marked by the arrows. The toe-prints at positions 61 (749T), 55 (755T), 54 (756T), 52 (758T) in the 3′ UTR are indicated. ( J ) Schematic representation of the toe-prints of PTB protein on putative predicted secondary structure of p53 3′UTR R-III (third region) RNA (nucleotides 759–809 in 3′UTR) generated by MFOLD. Nucleotides 748 to 758 bind PTB and are pointed out in red. The curly bracket region shows the miR-1285 binding site (seed sequence). ( K ) α- 32 P-labeled 3′UTR (wild type: WT, mutant: 2a = TT to GG 755–756, 3a = TT to GG 748–749) were UV crosslinked with increasing concentration of PTB (50 ng, 100 ng), lane 1 is no protein (NP). ( L ) H1299 cells were transfected with wild type Luc -3′UTR (labeled as WT), Luc -3′UTR Mut2a and Luc -3′UTR Mut3a (Mutant: 2a = TT to GG 755–756, 3a = TT to GG 748–749) and Rluc was used as transfection control. After 24 h, the cells were lysed and processed for luciferase activity. The graph shows the normalized Fluc activity (F/R) of different constructs ( n = 6).
    Figure Legend Snippet: Effect of knockdown of miRNAs on the expression of p53 isoforms and miRNAs interplay with PTB. ( A ) H1299 cells were co-transfected with Luc-3′UTR and anti-miRs for miR-30d/miR-1285/miR-504/miR-181 (10 nM) and Rluc (Renilla luciferase) as transfection control. After 48 h, the cells were lysed and processed for luciferase activity. The graph represents the normalized luciferase activity, i.e. Fluc/Rluc (F/R) ( n = 3). ( B ) H1299 cells were co-transfected with p53 cDNA construct having 3′UTR (5′UTR + cDNA + 3′UTR) or no 3′UTR (5′UTR + cDNA) and anti-miR-30d/anti-miR-1285 in two concentrations (10 nM and 20 nM). After 48 h, the cells were lysed and processed for immunoblotting. ( C ) Western blot analysis of cell extracts from A549-cells transfected with anti-miRs for miR-1285/miR-30d (30 nM), probed with CM1 after 48 h. Upper panel: p53 and Δ40p53; lower panel: actin. ( D ) Quantitative PCR of p21 and 14–3-3σ mRNA levels normalized to Actin in A549 cells transfected with anti-miRs for miR-1285/ miR-30d (30 nM) for 48 h ( n = 3). ( E ) H1299 cells were co-transfected with Luc-3′UTR and anti-miRs for miR-30d/miR-1285 (20 nM). After 48 h, the cells were lysed and RNP complexes were immunoprecipitated with anti-PTB antibody. RT PCR was performed for checking the levels of 3′UTR associated with PTB. The values for RNA immunoprecipitated with anti PTB antibody in different conditions (no anti-miR, anti-miR-30d and anti-miR-1285) were first normalized with its respective values for RNA immunoprecipitated with IgG control antibody and then the fold change was calculated using the value for 3′UTR pulled down with anti-PTB, without anti-miR treatment as the basal value ( n = 5). ( F ) H1299 cells were transfected with wild type Fluc -3′UTR (labeled as WT), Fluc -3′UTR mut miR-1285 M1 (3′UTR mutated for miR-1285 one binding site, labeled as M1), Fluc -3′UTR mut miR-1285 M1+M2 (3′UTR mutated for miR-1285 both binding sites, labeled as M1 + M2) and GFP expression from a GFP plasmid vector was used as transfection efficiency control. After 24 h, the cells were lysed and processed for luciferase activity. The graph shows normalized Fluc activity of different constructs ( n = 3). ( G ) H1299 cells were transfected with either wild type Fluc -3′UTR (labeled as WT) or Fluc -3′UTR mut miR-1285 M1+M2 (3′UTR mutated for miR-1285 both binding sites, labeled as M1 + M2). After 24 h, the cells were lysed and RNP complexes were immunoprecipitated with anti-PTB antibody. RT PCR was performed for checking the levels of 3′UTR associated with PTB. The values for RNA (WT and M1 + M2) immunoprecipitated with anti PTB antibody were first normalized with its respective values for RNA immunoprecipitated with IgG control antibody, and then the fold change was calculated using the value for 3′UTR pulled down with anti-PTB from WT-3′UTR transfected cells as the basal value ( n = 6). ( H ) H1299 cells were transfected with either wild type Fluc -3′UTR (labeled as WT) or Fluc -3′UTR mut miR-1285 M1+M2 (3′UTR mutated for miR-1285 both binding sites, labeled as M1 + M2). After 24 h, the cells were lysed and RNP complexes were immunoprecipitated with anti-Ago-2 antibody. q-RT PCR was performed for checking the levels of 3′UTR associated with Ago-2. The values for RNA (WT and M1+M2) immunoprecipitated with anti Ago-2 antibody were first normalized with its respective values for RNA immunoprecipitated with IgG control antibody and then the fold change was calculated using the value for 3′UTR pulled down with anti-Ago-2 from WT-3′UTR transfected cells as the basal value ( n = 6). ( I ) Toe-printing analysis to map the contact points of PTB on p53 3′UTR. p53 3′UTR RNA was incubated in absence (lane 7) and presence (lanes 5 and 6) of increasing concentrations of purified recombinant PTB (200 ng, 400 ng). The RNA in the ribonucleoprotein complexes were reverse transcribed using 3R3 reverse primer (p53 3′UTR region 3 reverse primer) and the resulting cDNAs were resolved in 8% acrylamide–8 M urea PAGE in parallel with a sequencing reaction. The cDNA products terminated at the sites due to protein binding is marked by the arrows. The toe-prints at positions 61 (749T), 55 (755T), 54 (756T), 52 (758T) in the 3′ UTR are indicated. ( J ) Schematic representation of the toe-prints of PTB protein on putative predicted secondary structure of p53 3′UTR R-III (third region) RNA (nucleotides 759–809 in 3′UTR) generated by MFOLD. Nucleotides 748 to 758 bind PTB and are pointed out in red. The curly bracket region shows the miR-1285 binding site (seed sequence). ( K ) α- 32 P-labeled 3′UTR (wild type: WT, mutant: 2a = TT to GG 755–756, 3a = TT to GG 748–749) were UV crosslinked with increasing concentration of PTB (50 ng, 100 ng), lane 1 is no protein (NP). ( L ) H1299 cells were transfected with wild type Luc -3′UTR (labeled as WT), Luc -3′UTR Mut2a and Luc -3′UTR Mut3a (Mutant: 2a = TT to GG 755–756, 3a = TT to GG 748–749) and Rluc was used as transfection control. After 24 h, the cells were lysed and processed for luciferase activity. The graph shows the normalized Fluc activity (F/R) of different constructs ( n = 6).

    Techniques Used: Expressing, Transfection, Luciferase, Activity Assay, Construct, Western Blot, Real-time Polymerase Chain Reaction, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Labeling, Binding Assay, Plasmid Preparation, Incubation, Purification, Recombinant, Polyacrylamide Gel Electrophoresis, Sequencing, Protein Binding, Generated, Mutagenesis, Concentration Assay

    5) Product Images from "Modulation of the colon cancer cell phenotype by pro-inflammatory macrophages: A preclinical model of surgery-associated inflammation and tumor recurrence"

    Article Title: Modulation of the colon cancer cell phenotype by pro-inflammatory macrophages: A preclinical model of surgery-associated inflammation and tumor recurrence

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0192958

    PMA-differentiated THP-1 and U937 macrophages displayed features of a M1 type phenotype. PMA-differentiated THP-1 and U937 macrophages ( MACRO ) were harvested in parallel with non-treated cultures ( MONO ), and total RNA isolated. Some cultures were further incubated in the presence of medroxyprogesterone ( MPA ) for 24 hours or left untreated ( MPA-CON ), and similarly harvested and total RNA isolated. RNA samples were reverse transcribed to cDNA, and Real Time PCR analysis was performed using oligonucleotide primers specific for the indicated genes (see Material and Methods ). Markers of the M1 phenotype ( IL6 , IL1B , CXCL10 ) displayed strong induction during PMA-differentiation but were abruptly suppressed with further incubation with the M2 inducer MPA. In contrast, induction of the M2 markers CD163 and IL10 was modest, and in the case of CD163 potently enhanced by the M2 inducer MPA. Results are shown as the relative levels of each mRNA in the differentiated cell lines ( MACRO , MPA-CON , MPA ) respect to the non-treated monocytic cell lines ( MONO ) and expressed as the mean ± error of at least 2 independent experiments.
    Figure Legend Snippet: PMA-differentiated THP-1 and U937 macrophages displayed features of a M1 type phenotype. PMA-differentiated THP-1 and U937 macrophages ( MACRO ) were harvested in parallel with non-treated cultures ( MONO ), and total RNA isolated. Some cultures were further incubated in the presence of medroxyprogesterone ( MPA ) for 24 hours or left untreated ( MPA-CON ), and similarly harvested and total RNA isolated. RNA samples were reverse transcribed to cDNA, and Real Time PCR analysis was performed using oligonucleotide primers specific for the indicated genes (see Material and Methods ). Markers of the M1 phenotype ( IL6 , IL1B , CXCL10 ) displayed strong induction during PMA-differentiation but were abruptly suppressed with further incubation with the M2 inducer MPA. In contrast, induction of the M2 markers CD163 and IL10 was modest, and in the case of CD163 potently enhanced by the M2 inducer MPA. Results are shown as the relative levels of each mRNA in the differentiated cell lines ( MACRO , MPA-CON , MPA ) respect to the non-treated monocytic cell lines ( MONO ) and expressed as the mean ± error of at least 2 independent experiments.

    Techniques Used: Isolation, Incubation, Real-time Polymerase Chain Reaction

    Gene expression analysis of EMT-related markers. The inflammatory medium conditioned by THP-1 or U937 macrophages induced the expression of several genes related to the EMT process. Total RNA was isolated from HT-29 or SW620 cell cultures that had been treated with the indicated conditioned media for 48 hours. RNA samples were reverse transcribed to cDNA, and Real Time PCR analysis was performed using oligonucleotide primers specific for the indicated genes (see Material and Methods ). Genes analyzed were: FN1 (fibronectin 1), VIM (vimentin), ITGB6 (integrin subunit beta 6), and S100A4 (S100 calcium binding protein A4). Results are shown as the relative levels of each mRNA respect to the sample treated with standard medium ( CON ) and expressed as the mean ± SD of at least 3 independent experiments. CON , standard medium; MONO-CM , conditioned medium from non-activated monocytes; MACRO-CM , conditioned medium from differentiated macrophages.
    Figure Legend Snippet: Gene expression analysis of EMT-related markers. The inflammatory medium conditioned by THP-1 or U937 macrophages induced the expression of several genes related to the EMT process. Total RNA was isolated from HT-29 or SW620 cell cultures that had been treated with the indicated conditioned media for 48 hours. RNA samples were reverse transcribed to cDNA, and Real Time PCR analysis was performed using oligonucleotide primers specific for the indicated genes (see Material and Methods ). Genes analyzed were: FN1 (fibronectin 1), VIM (vimentin), ITGB6 (integrin subunit beta 6), and S100A4 (S100 calcium binding protein A4). Results are shown as the relative levels of each mRNA respect to the sample treated with standard medium ( CON ) and expressed as the mean ± SD of at least 3 independent experiments. CON , standard medium; MONO-CM , conditioned medium from non-activated monocytes; MACRO-CM , conditioned medium from differentiated macrophages.

    Techniques Used: Expressing, Isolation, Real-time Polymerase Chain Reaction, Binding Assay

    Gene expression analysis of β-catenin/TCF4 transcriptional target genes. The inflammatory medium conditioned by THP-1 or U937 macrophages induced the expression of some targets of the β-catenin/TCF4 transcriptional pathway. Total RNA was isolated from HT-29 or SW620 cell cultures that had been treated with the indicated conditioned media for 48 hours. RNA samples were reverse transcribed to cDNA, and Real Time PCR analysis was performed using oligonucleotide primers specific for the indicated genes (see Material and Methods ). Genes analyzed were: MMP7 (matrix metallopeptidase 7), PTGS2 (prostaglandin-endoperoxide synthase 2, or COX-2), MET (MET proto-oncogene, receptor tyrosine kinase), and CCND1 (cyclin D1). Results are shown as the relative levels of each mRNA respect to the sample treated with standard medium ( CON ) and expressed as the mean ± SD of at least 3 independent experiments. CON , standard medium; MONO-CM , conditioned medium from non-activated monocytes; MACRO-CM , conditioned medium from differentiated macrophages.
    Figure Legend Snippet: Gene expression analysis of β-catenin/TCF4 transcriptional target genes. The inflammatory medium conditioned by THP-1 or U937 macrophages induced the expression of some targets of the β-catenin/TCF4 transcriptional pathway. Total RNA was isolated from HT-29 or SW620 cell cultures that had been treated with the indicated conditioned media for 48 hours. RNA samples were reverse transcribed to cDNA, and Real Time PCR analysis was performed using oligonucleotide primers specific for the indicated genes (see Material and Methods ). Genes analyzed were: MMP7 (matrix metallopeptidase 7), PTGS2 (prostaglandin-endoperoxide synthase 2, or COX-2), MET (MET proto-oncogene, receptor tyrosine kinase), and CCND1 (cyclin D1). Results are shown as the relative levels of each mRNA respect to the sample treated with standard medium ( CON ) and expressed as the mean ± SD of at least 3 independent experiments. CON , standard medium; MONO-CM , conditioned medium from non-activated monocytes; MACRO-CM , conditioned medium from differentiated macrophages.

    Techniques Used: Expressing, Isolation, Real-time Polymerase Chain Reaction

    Cytokine genes induced by PMA-differentiated THP-1 and U937 macrophages suggested common mechanisms with the tumor recurrence in colorectal cancer patients undergoing postoperative peritoneal infections. PMA-differentiated THP-1 and U937 macrophages (MACRO) were harvested in parellel with non-treated cultures (MONO), and total RNA isolated. RNA samples were reverse transcribed to cDNA, and Real Time PCR analysis was performed using oligonucleotide primers specific for the indicated genes (see Material and Methods ). Genes analyzed corresponded to a gene panel differentially expressed in peripheral blood leukocytes of colorectal cancer patients undergoing postoperative peritoneal infetions (see reference 14 ): MMP9 (matrix metallopeptidase 9), IL1R2 (interleukin 1 receptor type 2), PLSCR1 (phospholipid scramblase 1), TRPS1 (transcriptional repressor GATA binding 1), ORM1 (orosomucoid 1), HP (haptoglobin), ADAM9 (ADAM metallopeptidase domain 9), and PDGFC (platelet derived growth factor C). Results are shown as the relative levels of each mRNA in the PMA treated cell lines ( MACRO ) respect to the non-treated monocytic cell lines ( MONO ) and expressed as the mean ± SD of at least 3 independent experiments. N . D ., not detectable.
    Figure Legend Snippet: Cytokine genes induced by PMA-differentiated THP-1 and U937 macrophages suggested common mechanisms with the tumor recurrence in colorectal cancer patients undergoing postoperative peritoneal infections. PMA-differentiated THP-1 and U937 macrophages (MACRO) were harvested in parellel with non-treated cultures (MONO), and total RNA isolated. RNA samples were reverse transcribed to cDNA, and Real Time PCR analysis was performed using oligonucleotide primers specific for the indicated genes (see Material and Methods ). Genes analyzed corresponded to a gene panel differentially expressed in peripheral blood leukocytes of colorectal cancer patients undergoing postoperative peritoneal infetions (see reference 14 ): MMP9 (matrix metallopeptidase 9), IL1R2 (interleukin 1 receptor type 2), PLSCR1 (phospholipid scramblase 1), TRPS1 (transcriptional repressor GATA binding 1), ORM1 (orosomucoid 1), HP (haptoglobin), ADAM9 (ADAM metallopeptidase domain 9), and PDGFC (platelet derived growth factor C). Results are shown as the relative levels of each mRNA in the PMA treated cell lines ( MACRO ) respect to the non-treated monocytic cell lines ( MONO ) and expressed as the mean ± SD of at least 3 independent experiments. N . D ., not detectable.

    Techniques Used: Isolation, Real-time Polymerase Chain Reaction, Binding Assay, Derivative Assay

    Gene expression analysis of EMT-related transcription factors. The inflammatory medium conditioned by THP-1 or U937 macrophages induced the expression of some transcription factor genes known as mediators of the EMT process. Total RNA was isolated from HT-29 or SW620 cell cultures that had been treated with the indicated conditioned media for 48 hours. RNA samples were reverse transcribed to cDNA, and Real Time PCR analysis was performed using oligonucleotide primers specific for the indicated genes (see Material and Methods ). Genes analyzed were: ZEB1 (zinc finger E-box binding homeobox 1), SNAI1 (snail family transcriptional repressor 1, or Snail), and SNAI2 (snail family transcriptional repressor 2, or Slug). Results are shown as the relative levels of each mRNA respect to the sample treated with standard medium ( CON ) and expressed as the mean ± SD of at least 3 independent experiments. N . D ., not detectable; CON , standard medium; MONO-CM , conditioned medium from non-activated monocytes; MACRO-CM , conditioned medium from differentiated macrophages.
    Figure Legend Snippet: Gene expression analysis of EMT-related transcription factors. The inflammatory medium conditioned by THP-1 or U937 macrophages induced the expression of some transcription factor genes known as mediators of the EMT process. Total RNA was isolated from HT-29 or SW620 cell cultures that had been treated with the indicated conditioned media for 48 hours. RNA samples were reverse transcribed to cDNA, and Real Time PCR analysis was performed using oligonucleotide primers specific for the indicated genes (see Material and Methods ). Genes analyzed were: ZEB1 (zinc finger E-box binding homeobox 1), SNAI1 (snail family transcriptional repressor 1, or Snail), and SNAI2 (snail family transcriptional repressor 2, or Slug). Results are shown as the relative levels of each mRNA respect to the sample treated with standard medium ( CON ) and expressed as the mean ± SD of at least 3 independent experiments. N . D ., not detectable; CON , standard medium; MONO-CM , conditioned medium from non-activated monocytes; MACRO-CM , conditioned medium from differentiated macrophages.

    Techniques Used: Expressing, Isolation, Real-time Polymerase Chain Reaction, Binding Assay

    6) Product Images from "Impact of MUC1 Mucin Downregulation in the Phenotypic Characteristics of MKN45 Gastric Carcinoma Cell Line"

    Article Title: Impact of MUC1 Mucin Downregulation in the Phenotypic Characteristics of MKN45 Gastric Carcinoma Cell Line

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0026970

    MUC1 downregulation by shRNA. ( A ) MUC1 detection by immunofluorescence with HMFG-1 antibody in MKN45-C1 and MKN45-C2 and MKN45-SC control; ( B ) MUC1 protein detection by western-blot with MUC1-Ab5 antibody of total protein extracts from MKN45-C1 and MKN45-C2 and MKN45-SC control; ( C ) Quantification of MUC1 RNA in MKN45-C1 and MKN45-C2 and MKN45-SC control by real-time PCR. MUC1 expression was corrected to the house-keeping gene 18S and normalized to the data obtained with the scrambled control. *P
    Figure Legend Snippet: MUC1 downregulation by shRNA. ( A ) MUC1 detection by immunofluorescence with HMFG-1 antibody in MKN45-C1 and MKN45-C2 and MKN45-SC control; ( B ) MUC1 protein detection by western-blot with MUC1-Ab5 antibody of total protein extracts from MKN45-C1 and MKN45-C2 and MKN45-SC control; ( C ) Quantification of MUC1 RNA in MKN45-C1 and MKN45-C2 and MKN45-SC control by real-time PCR. MUC1 expression was corrected to the house-keeping gene 18S and normalized to the data obtained with the scrambled control. *P

    Techniques Used: shRNA, Immunofluorescence, Western Blot, Real-time Polymerase Chain Reaction, Expressing

    7) Product Images from "A novel FLI1 exonic circular RNA promotes metastasis in breast cancer by coordinately regulating TET1 and DNMT1"

    Article Title: A novel FLI1 exonic circular RNA promotes metastasis in breast cancer by coordinately regulating TET1 and DNMT1

    Journal: Genome Biology

    doi: 10.1186/s13059-018-1594-y

    FECR1 upregulates FLI1 . a Diagram of the RNA reverse transcription-associated trap (RAT) assay. FECR1 was in situ reverse transcribed using circular RNA-specific primers in the presence of biotin-dCTP. The FECR1-interacting chromatin DNAs were isolated for library sequencing. b Location of PCR primers to detect the interaction of FECR1 at the FLI1 locus. 5′-CT, 5′-upstream control site. c Binding of FECR1 in the FLI1 locus. The FECR1 RAT-captured chromatin DNAs were amplified by PCR using primers covering the FLI1 locus. Note the binding of FECR1 in the FLI1 promoter (P1, P2). d Activation of FLI1 by FECR1. Expression of FLI1 was quantitated by qPCR using two pairs of primers that cover different regions of FLI1 . Region 1, the PCR product covers exon 4 to exon 6; region 2, the PCR product covers exon 3 to exon 4. ** p
    Figure Legend Snippet: FECR1 upregulates FLI1 . a Diagram of the RNA reverse transcription-associated trap (RAT) assay. FECR1 was in situ reverse transcribed using circular RNA-specific primers in the presence of biotin-dCTP. The FECR1-interacting chromatin DNAs were isolated for library sequencing. b Location of PCR primers to detect the interaction of FECR1 at the FLI1 locus. 5′-CT, 5′-upstream control site. c Binding of FECR1 in the FLI1 locus. The FECR1 RAT-captured chromatin DNAs were amplified by PCR using primers covering the FLI1 locus. Note the binding of FECR1 in the FLI1 promoter (P1, P2). d Activation of FLI1 by FECR1. Expression of FLI1 was quantitated by qPCR using two pairs of primers that cover different regions of FLI1 . Region 1, the PCR product covers exon 4 to exon 6; region 2, the PCR product covers exon 3 to exon 4. ** p

    Techniques Used: In Situ, Isolation, Sequencing, Polymerase Chain Reaction, Binding Assay, Amplification, Activation Assay, Expressing, Real-time Polymerase Chain Reaction

    FLI1 circular RNA promotes invasion of breast cancer cells. a Ectopic expression of FECR1 in MDA-MB231 breast cancer cells. The FECR1 expression cassette is composed of FLI1 exons 4-2-3 and the intron fragments containing the back-splicing elements. pCMV, CMV promoter; pEF1a, EF1a promoter. DsRed fluorescent marker was used to track the transfection. b RT-PCR of FECR1. After stable transfection, cells were collected and FECR1 was amplified by PCR. PBS and vector, control groups. c Quantitation of FECR1 by qPCR. ** p
    Figure Legend Snippet: FLI1 circular RNA promotes invasion of breast cancer cells. a Ectopic expression of FECR1 in MDA-MB231 breast cancer cells. The FECR1 expression cassette is composed of FLI1 exons 4-2-3 and the intron fragments containing the back-splicing elements. pCMV, CMV promoter; pEF1a, EF1a promoter. DsRed fluorescent marker was used to track the transfection. b RT-PCR of FECR1. After stable transfection, cells were collected and FECR1 was amplified by PCR. PBS and vector, control groups. c Quantitation of FECR1 by qPCR. ** p

    Techniques Used: Expressing, Multiple Displacement Amplification, Marker, Transfection, Reverse Transcription Polymerase Chain Reaction, Stable Transfection, Amplification, Polymerase Chain Reaction, Plasmid Preparation, Quantitation Assay, Real-time Polymerase Chain Reaction

    8) Product Images from "Visfatin Induces Sickness Responses in the Brain"

    Article Title: Visfatin Induces Sickness Responses in the Brain

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0015981

    Visfatin-induced increases in hypothalamic mRNA levels of pro-inflammatory cytokines and prostaglandin-synthesizing enzymes. RNA was extracted from rat hypothalami 6 h after ICV injection of visfatin. mRNA expressions encoding TNF-α, IL-1β, COX2 and mPGES-1 were determined using real-time PCR. Visfatin significantly stimulated the expressions of TNF-α (A), IL-1β (B), COX2 (C) and mPGES-1 (D) mRNA in the hypothalamus. Data are represented as mean ± SEM (n = 6). **P
    Figure Legend Snippet: Visfatin-induced increases in hypothalamic mRNA levels of pro-inflammatory cytokines and prostaglandin-synthesizing enzymes. RNA was extracted from rat hypothalami 6 h after ICV injection of visfatin. mRNA expressions encoding TNF-α, IL-1β, COX2 and mPGES-1 were determined using real-time PCR. Visfatin significantly stimulated the expressions of TNF-α (A), IL-1β (B), COX2 (C) and mPGES-1 (D) mRNA in the hypothalamus. Data are represented as mean ± SEM (n = 6). **P

    Techniques Used: Injection, Real-time Polymerase Chain Reaction

    9) Product Images from "Curcumin attenuates the effects of insulin on stimulating hepatic stellate cell activation by interrupting insulin signaling and attenuating oxidative stress"

    Article Title: Curcumin attenuates the effects of insulin on stimulating hepatic stellate cell activation by interrupting insulin signaling and attenuating oxidative stress

    Journal: Laboratory investigation; a journal of technical methods and pathology

    doi: 10.1038/labinvest.2009.115

    Curcumin eliminates the effect of insulin and induces gene expression of GCLc and GCLm in activated HSCs in vitro Serum-starved HSCs were stimulated with insulin at indicated concentrations in the absence ( A B ) or presence ( C D ) of curcumin at 0–30μM in serum-free media for 24 hr. Total RNA or whole cell extracts were prepared for real-time PCR assays ( A C ), or Western blotting analyses ( B D ). β-actin was used as an invariant internal control for calculating mRNA fold changes (n=3). * p
    Figure Legend Snippet: Curcumin eliminates the effect of insulin and induces gene expression of GCLc and GCLm in activated HSCs in vitro Serum-starved HSCs were stimulated with insulin at indicated concentrations in the absence ( A B ) or presence ( C D ) of curcumin at 0–30μM in serum-free media for 24 hr. Total RNA or whole cell extracts were prepared for real-time PCR assays ( A C ), or Western blotting analyses ( B D ). β-actin was used as an invariant internal control for calculating mRNA fold changes (n=3). * p

    Techniques Used: Expressing, In Vitro, Real-time Polymerase Chain Reaction, Western Blot

    de novo synthesis of GSH is required for curcumin to suppress gene expression of InsR and other proteins relevant to HSC activation induced by insulin Serum-starved HSCs were divided into two groups. One group of cells was treated with or without insulin (100 nM) plus curcumin (Cur) (20 μM) or NAC (5 mM) in serum-free media for 24 hr. Another group of cells was pretreated with BSO (0.25 mM) for 1 hr prior to the addition of insulin (100 nM) plus curcumin (20 μM) or NAC (5 mM) in serum-free media for additional 24 hr. Total RNA or whole cell extracts were respectively prepared for real-time PCR assays ( A ) or Western blotting analyses ( B ). β-actin was used as an invariant internal control for calculating mRNA fold changes (n=3). * p
    Figure Legend Snippet: de novo synthesis of GSH is required for curcumin to suppress gene expression of InsR and other proteins relevant to HSC activation induced by insulin Serum-starved HSCs were divided into two groups. One group of cells was treated with or without insulin (100 nM) plus curcumin (Cur) (20 μM) or NAC (5 mM) in serum-free media for 24 hr. Another group of cells was pretreated with BSO (0.25 mM) for 1 hr prior to the addition of insulin (100 nM) plus curcumin (20 μM) or NAC (5 mM) in serum-free media for additional 24 hr. Total RNA or whole cell extracts were respectively prepared for real-time PCR assays ( A ) or Western blotting analyses ( B ). β-actin was used as an invariant internal control for calculating mRNA fold changes (n=3). * p

    Techniques Used: Expressing, Activation Assay, Real-time Polymerase Chain Reaction, Western Blot

    Curcumin dose-dependently suppresses gene expression of InsR in cultured HSCs Serum-starved HSCs were stimulated with or without insulin (100 nM) in the presence or absence of curcumin at indicated concentrations in serum-free media for 24 hr. Total RNA or whole cell extracts were prepared. ( A ). Real-time PCR assays of the steady state level of InsR mRNA. β-actin was used as an invariant internal control for calculating mRNA fold changes (n=3). * p
    Figure Legend Snippet: Curcumin dose-dependently suppresses gene expression of InsR in cultured HSCs Serum-starved HSCs were stimulated with or without insulin (100 nM) in the presence or absence of curcumin at indicated concentrations in serum-free media for 24 hr. Total RNA or whole cell extracts were prepared. ( A ). Real-time PCR assays of the steady state level of InsR mRNA. β-actin was used as an invariant internal control for calculating mRNA fold changes (n=3). * p

    Techniques Used: Expressing, Cell Culture, Real-time Polymerase Chain Reaction

    Curcumin attenuates the stimulatory effects of insulin on the activation of HSCs Serum-starved HSCs were stimulated with or without insulin (100 nM) plus curcumin at various concentrations in serum-depleted DMEM for 24 hr. Total RNA or whole cell extracts were prepared for real-time PCR assays ( A C ), or for Western blotting analyses ( B D ). Values in A C were presented as mRNA fold changes (mean ± S. D., n=3), * p
    Figure Legend Snippet: Curcumin attenuates the stimulatory effects of insulin on the activation of HSCs Serum-starved HSCs were stimulated with or without insulin (100 nM) plus curcumin at various concentrations in serum-depleted DMEM for 24 hr. Total RNA or whole cell extracts were prepared for real-time PCR assays ( A C ), or for Western blotting analyses ( B D ). Values in A C were presented as mRNA fold changes (mean ± S. D., n=3), * p

    Techniques Used: Activation Assay, Real-time Polymerase Chain Reaction, Western Blot

    10) Product Images from "IL-17 producing innate lymphoid cells and the NLRP3 inflammasome facilitate obesity-associated airway hyperreactivity"

    Article Title: IL-17 producing innate lymphoid cells and the NLRP3 inflammasome facilitate obesity-associated airway hyperreactivity

    Journal: Nature medicine

    doi: 10.1038/nm.3423

    IL-1β production and M1 macrophages are increased in the lungs of obese mice (a–f). Total RNA was extracted from the lung and adipose tissue for qRT-PCR. Fold induction of Il1b ( a,d ), Il6 ( b,e ), and Il23 ( c,f ) were calculated based on GAPDH expression. * p
    Figure Legend Snippet: IL-1β production and M1 macrophages are increased in the lungs of obese mice (a–f). Total RNA was extracted from the lung and adipose tissue for qRT-PCR. Fold induction of Il1b ( a,d ), Il6 ( b,e ), and Il23 ( c,f ) were calculated based on GAPDH expression. * p

    Techniques Used: Mouse Assay, Quantitative RT-PCR, Expressing

    11) Product Images from "Translation of CircRNAs"

    Article Title: Translation of CircRNAs

    Journal: Molecular Cell

    doi: 10.1016/j.molcel.2017.02.021

    CircRNAs Can Produce Proteins as Tagged Minigenes (A) Ribo-circRNA tagging strategy. MT, metallothionein promoter. (B) circMbl V5, Pde8 V5, and Cdi V5 minigenes produce proteins of their expected size (red arrows). GFP was co-transfected. (C) circMbl minigenes produce both circMbl and linear concatemers when transfected into Drosophila S2 cells. Left: EtBr staining. Right: northern blot using a probe directed against the circMbl backsplice junction. (D) Top: scheme of the minigene expressing a split Cherry molecule under the control of the circMbl cUTR. Bottom: representative picture of Drosophila S2 cells transfected with this minigene. (E) Western blot from Drosophila S2 cells transfected with the specified circMbl V5 minigenes. (F) Northern blot using a probe directed against the circMbl backsplice junction. Samples were prepared from heads of control ( actin-gal4 /+) or circMbl overexpression (OE) flies ( actin-gal4 ; UAS-circMbl). Left: EtBr staining. Right: northern blot. The asterisk indicates an unknown RNA species that is detected by the probe, and it is resistant to RNaseR. (G) RT-PCR analysis of control and circMbl OE flies. Gene expression was normalized to rp49 and 28S RNAs. Mean ± SD (n = 2 for control and n = 3 for circMbl samples). (H) Western blot of control or circMbl OE flies utilizing the anti-MBL or anti- tubulin antibodies. See Figures S1 and S2 .
    Figure Legend Snippet: CircRNAs Can Produce Proteins as Tagged Minigenes (A) Ribo-circRNA tagging strategy. MT, metallothionein promoter. (B) circMbl V5, Pde8 V5, and Cdi V5 minigenes produce proteins of their expected size (red arrows). GFP was co-transfected. (C) circMbl minigenes produce both circMbl and linear concatemers when transfected into Drosophila S2 cells. Left: EtBr staining. Right: northern blot using a probe directed against the circMbl backsplice junction. (D) Top: scheme of the minigene expressing a split Cherry molecule under the control of the circMbl cUTR. Bottom: representative picture of Drosophila S2 cells transfected with this minigene. (E) Western blot from Drosophila S2 cells transfected with the specified circMbl V5 minigenes. (F) Northern blot using a probe directed against the circMbl backsplice junction. Samples were prepared from heads of control ( actin-gal4 /+) or circMbl overexpression (OE) flies ( actin-gal4 ; UAS-circMbl). Left: EtBr staining. Right: northern blot. The asterisk indicates an unknown RNA species that is detected by the probe, and it is resistant to RNaseR. (G) RT-PCR analysis of control and circMbl OE flies. Gene expression was normalized to rp49 and 28S RNAs. Mean ± SD (n = 2 for control and n = 3 for circMbl samples). (H) Western blot of control or circMbl OE flies utilizing the anti-MBL or anti- tubulin antibodies. See Figures S1 and S2 .

    Techniques Used: Transfection, Staining, Northern Blot, Expressing, Western Blot, Over Expression, Reverse Transcription Polymerase Chain Reaction

    12) Product Images from "Utilization of Rad51C promoter for transcriptional targeting of cancer cells"

    Article Title: Utilization of Rad51C promoter for transcriptional targeting of cancer cells

    Journal: Oncotarget

    doi:

    Rad51C transcripts are upregulated in cancer cells A. Quantitative analysis of mRNA expression of Rad51B, Rad51C, Rad51D and Rad52 in normal and cancer cells. Exponentially growing cells were harvested for total RNA extraction. The transcript levels were determined using real time RT-PCR followed by analysis using delta CT method [ 29 ]. Then the relative expression level of Rad genes in different types of cells was normalized to that in HCA2 cells. All experiments were repeated at least 9 times. Error bars represent s.d. B. Statistical analysis of expression of Rad51B, Rad51C, Rad51D and Rad52 in normal and cancer cells. Man Whitney U test was employed to examine significance. * The upregulation of Rad51C in cancer cells was statistically significant ( P MWU =0.037).
    Figure Legend Snippet: Rad51C transcripts are upregulated in cancer cells A. Quantitative analysis of mRNA expression of Rad51B, Rad51C, Rad51D and Rad52 in normal and cancer cells. Exponentially growing cells were harvested for total RNA extraction. The transcript levels were determined using real time RT-PCR followed by analysis using delta CT method [ 29 ]. Then the relative expression level of Rad genes in different types of cells was normalized to that in HCA2 cells. All experiments were repeated at least 9 times. Error bars represent s.d. B. Statistical analysis of expression of Rad51B, Rad51C, Rad51D and Rad52 in normal and cancer cells. Man Whitney U test was employed to examine significance. * The upregulation of Rad51C in cancer cells was statistically significant ( P MWU =0.037).

    Techniques Used: Expressing, RNA Extraction, Quantitative RT-PCR

    13) Product Images from "Fasciola hepatica glycoconjugates immuneregulate dendritic cells through the Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin inducing T cell anergy"

    Article Title: Fasciola hepatica glycoconjugates immuneregulate dendritic cells through the Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin inducing T cell anergy

    Journal: Scientific Reports

    doi: 10.1038/srep46748

    F. hepatica glycoconjugates favor the production of IL-10 by TLR-triggered mo-DCs. ( A ) IL-6, IL-10, TNFα and IL-12p70 levels determined by ELISA on supernatants from Pam3CSK4- and LPS-stimulated mo-DC cultures incubated with and without FhTE. ( B ) IL-10, IL-27p28, IL-27 EBI3 and IL-12p35 levels determined by qRT-PCR of purified RNA of Pam3CSK4- and LPS-sitmulated mo-DC cultures incubated with and without FhTE. ( C ) IL-10 levels determined by ELISA on supernatants of TLR-triggered mo-DCs incubated in the presence of FhTE, FhCB (oxidation negative control) or FhmPox (oxidized FhTE). A representative Figure of four independent experiments is shown (±SD, indicated by error bars). Asterisks indicate statistically significant differences ( p
    Figure Legend Snippet: F. hepatica glycoconjugates favor the production of IL-10 by TLR-triggered mo-DCs. ( A ) IL-6, IL-10, TNFα and IL-12p70 levels determined by ELISA on supernatants from Pam3CSK4- and LPS-stimulated mo-DC cultures incubated with and without FhTE. ( B ) IL-10, IL-27p28, IL-27 EBI3 and IL-12p35 levels determined by qRT-PCR of purified RNA of Pam3CSK4- and LPS-sitmulated mo-DC cultures incubated with and without FhTE. ( C ) IL-10 levels determined by ELISA on supernatants of TLR-triggered mo-DCs incubated in the presence of FhTE, FhCB (oxidation negative control) or FhmPox (oxidized FhTE). A representative Figure of four independent experiments is shown (±SD, indicated by error bars). Asterisks indicate statistically significant differences ( p

    Techniques Used: Enzyme-linked Immunosorbent Assay, Incubation, Quantitative RT-PCR, Purification, Negative Control

    14) Product Images from "Enterovirus 71 infection of human airway organoids reveals VP1-145 as a viral infectivity determinant"

    Article Title: Enterovirus 71 infection of human airway organoids reveals VP1-145 as a viral infectivity determinant

    Journal: Emerging Microbes & Infections

    doi: 10.1038/s41426-018-0077-2

    Replication kinetics of EV71 strains with a glutamic acid (E), glycine (G), or glutamine (Q) at VP1-145, generated by site-directed mutagenesis, in human airway organoids. a Fold increases in viral RNA of C1, C2, and B3 strains with VP1-145E, -G, or -Q in donor N41, determined by RT-PCR assay on lysed organoids. Data present the mean fold increase in two infection experiments + SEM. b Viral titers detected in the medium covering embedded organoids at 0–72 h postinfection. Titers are expressed as the mean 50% cell culture infective dose (CCID 50 )/ml in two infection experiments+SEM
    Figure Legend Snippet: Replication kinetics of EV71 strains with a glutamic acid (E), glycine (G), or glutamine (Q) at VP1-145, generated by site-directed mutagenesis, in human airway organoids. a Fold increases in viral RNA of C1, C2, and B3 strains with VP1-145E, -G, or -Q in donor N41, determined by RT-PCR assay on lysed organoids. Data present the mean fold increase in two infection experiments + SEM. b Viral titers detected in the medium covering embedded organoids at 0–72 h postinfection. Titers are expressed as the mean 50% cell culture infective dose (CCID 50 )/ml in two infection experiments+SEM

    Techniques Used: Generated, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Infection, Cell Culture

    Replication kinetics of enterovirus 71 (EV71) in human airway organoids. a , b Fold increases in viral RNA of EV71 subgenotype strains C1 (C1 91-480), C2 (2485), C4 (75-Yamagata), C5 (209-VN), B3 (SK-EV006), and B4 (C7-Osaka) in donors N39 and N41, determined by RT-PCR on lysed organoids. Data present the mean fold increase in two infection experiments + standard error of the mean (SEM). c Viral titers detected in the medium covering the gel-embedded organoids at 72 h postinfection (p.i.). Titers are expressed as the mean 50% cell culture infective dose (CCID 50 )/ml in two infection experiments + SEM
    Figure Legend Snippet: Replication kinetics of enterovirus 71 (EV71) in human airway organoids. a , b Fold increases in viral RNA of EV71 subgenotype strains C1 (C1 91-480), C2 (2485), C4 (75-Yamagata), C5 (209-VN), B3 (SK-EV006), and B4 (C7-Osaka) in donors N39 and N41, determined by RT-PCR on lysed organoids. Data present the mean fold increase in two infection experiments + standard error of the mean (SEM). c Viral titers detected in the medium covering the gel-embedded organoids at 72 h postinfection (p.i.). Titers are expressed as the mean 50% cell culture infective dose (CCID 50 )/ml in two infection experiments + SEM

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Infection, Cell Culture

    Replication kinetics of EV71 subgenotype C1 strains with a glutamine (Q) or glutamic acid (E) at VP1 residue 145 in human airway organoids. a , b Fold increases in viral RNA of EV71 strains in donors N39 and N41, respectively. Values presented were calculated from RT-PCR assays of lysed organoids. c , d Viral titers detected in the medium covering embedded organoids of donors N39 and N41, respectively, at 0 to 72 h postinfection. Titers are expressed as the 50% cell culture infective dose (CCID 50 )/ml
    Figure Legend Snippet: Replication kinetics of EV71 subgenotype C1 strains with a glutamine (Q) or glutamic acid (E) at VP1 residue 145 in human airway organoids. a , b Fold increases in viral RNA of EV71 strains in donors N39 and N41, respectively. Values presented were calculated from RT-PCR assays of lysed organoids. c , d Viral titers detected in the medium covering embedded organoids of donors N39 and N41, respectively, at 0 to 72 h postinfection. Titers are expressed as the 50% cell culture infective dose (CCID 50 )/ml

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Cell Culture

    15) Product Images from "Epigenetic targeting of bromodomain protein BRD4 counteracts cancer cachexia and prolongs survival"

    Article Title: Epigenetic targeting of bromodomain protein BRD4 counteracts cancer cachexia and prolongs survival

    Journal: Nature Communications

    doi: 10.1038/s41467-017-01645-7

    JQ1 administration blocks epididymal adipose tissue wasting. a Representative western blot and densitometric analysis of ATGL expression in epididymal adipose tissue (eAT) from control animals and C26-tumor-bearing mice treated with vehicle, (−)-JQ1, and (+)-JQ1 (animals per group: n = 3). Data represent means ± SD. b Representative western blot and densitometric analysis of the nuclear and transcriptionally active fragment of SREBP-1 (nSREBP-1) in the eAT of control and C26-tumor-bearing mice treated with vehicle, (−)-JQ1, and ( +)-JQ1. Four animals were used for each experimental condition. Data represent means ± SD. c , d Total RNA was extracted from eAT of control and C26-tumor-bearing mice (animals per group: n = 5) treated with vehicle or JQ1 (−/+) and expression levels of ACCα and FAS were measured by quantitative RT-PCR. Data represent means ± SD. e Representative western blot and densitometric analysis of p-AMPK (Thr172) in eAT of the six experimental groups described in a . Four mice were used for each experimental group. Data represent means ± SD. f Representative western blot and densitometric analysis of ACCα protein expression and p-ACCα inhibitory phosphorylation (Ser79) in eAT of control and C26 tumor-bearing mice (animals per group: n = 4) treated with vehicle or JQ1 (−/+). Data represent means ± SD. Statistical analysis was performed by using one-way ANOVA followed by Tukey’s post hoc test. * p
    Figure Legend Snippet: JQ1 administration blocks epididymal adipose tissue wasting. a Representative western blot and densitometric analysis of ATGL expression in epididymal adipose tissue (eAT) from control animals and C26-tumor-bearing mice treated with vehicle, (−)-JQ1, and (+)-JQ1 (animals per group: n = 3). Data represent means ± SD. b Representative western blot and densitometric analysis of the nuclear and transcriptionally active fragment of SREBP-1 (nSREBP-1) in the eAT of control and C26-tumor-bearing mice treated with vehicle, (−)-JQ1, and ( +)-JQ1. Four animals were used for each experimental condition. Data represent means ± SD. c , d Total RNA was extracted from eAT of control and C26-tumor-bearing mice (animals per group: n = 5) treated with vehicle or JQ1 (−/+) and expression levels of ACCα and FAS were measured by quantitative RT-PCR. Data represent means ± SD. e Representative western blot and densitometric analysis of p-AMPK (Thr172) in eAT of the six experimental groups described in a . Four mice were used for each experimental group. Data represent means ± SD. f Representative western blot and densitometric analysis of ACCα protein expression and p-ACCα inhibitory phosphorylation (Ser79) in eAT of control and C26 tumor-bearing mice (animals per group: n = 4) treated with vehicle or JQ1 (−/+). Data represent means ± SD. Statistical analysis was performed by using one-way ANOVA followed by Tukey’s post hoc test. * p

    Techniques Used: Western Blot, Expressing, Mouse Assay, Quantitative RT-PCR

    Muscle proteolysis and autophagy are hindered by JQ1 administration. a Representative immunoblot showing fast myosin heavy chain (MyHC) protein expression in TA whole extracts from the six animal groups. Upper panel shows quantification of immunoblot bands from four animals per group. Data represent means ± SD. b TA extracts from control and C26-tumor-bearing mice (animals per group: n = 3) were used in an in vitro degradation assay to reveal fast MyHC degradation in control vs. tumor-bearing mice treated with vehicle or JQ1 (−/+). c , d Total RNA was extracted from TA muscles from control and C26-tumor-bearing mice (animals per group: n = 10) treated with vehicle or JQ1 (−/+) and expression levels of MuRF1, MAFbx/Atrogin-1 were measured by quantitative RT-PCR. Data represent means ± SD. e Representative western blot of MAFbx/Atrogin-1 on TA whole extracts (animals per group: n = 4). Bands quantifications are shown in the upper panel. Data represent means ± SD. f – h Quantitative RT-PCR of autophagy genes (Bnip3, GABARAPL1, Cathepsin L) from control and C26-tumor-bearing mice. Ten animals were used for each experimental group. Data represent means ± SD. i , j Representative western blot for LC3 and p-Ulk1 in TA extracts of control and C26-tumor-bearing mice (animals per group: n = 4). Upper panel: quantification of normalized band intensity. Data represent means ± SD. Statistical analysis was performed by using one-way ANOVA followed by Tukey’s post hoc test. * p
    Figure Legend Snippet: Muscle proteolysis and autophagy are hindered by JQ1 administration. a Representative immunoblot showing fast myosin heavy chain (MyHC) protein expression in TA whole extracts from the six animal groups. Upper panel shows quantification of immunoblot bands from four animals per group. Data represent means ± SD. b TA extracts from control and C26-tumor-bearing mice (animals per group: n = 3) were used in an in vitro degradation assay to reveal fast MyHC degradation in control vs. tumor-bearing mice treated with vehicle or JQ1 (−/+). c , d Total RNA was extracted from TA muscles from control and C26-tumor-bearing mice (animals per group: n = 10) treated with vehicle or JQ1 (−/+) and expression levels of MuRF1, MAFbx/Atrogin-1 were measured by quantitative RT-PCR. Data represent means ± SD. e Representative western blot of MAFbx/Atrogin-1 on TA whole extracts (animals per group: n = 4). Bands quantifications are shown in the upper panel. Data represent means ± SD. f – h Quantitative RT-PCR of autophagy genes (Bnip3, GABARAPL1, Cathepsin L) from control and C26-tumor-bearing mice. Ten animals were used for each experimental group. Data represent means ± SD. i , j Representative western blot for LC3 and p-Ulk1 in TA extracts of control and C26-tumor-bearing mice (animals per group: n = 4). Upper panel: quantification of normalized band intensity. Data represent means ± SD. Statistical analysis was performed by using one-way ANOVA followed by Tukey’s post hoc test. * p

    Techniques Used: Expressing, Mouse Assay, In Vitro, Degradation Assay, Quantitative RT-PCR, Western Blot

    16) Product Images from "The Transcription and Translation Landscapes during Human Cytomegalovirus Infection Reveal Novel Host-Pathogen Interactions"

    Article Title: The Transcription and Translation Landscapes during Human Cytomegalovirus Infection Reveal Novel Host-Pathogen Interactions

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1005288

    Integration between ribosome footprints and protein abundance allows detection of immune ligands that are degraded during infection. A. Ribosome profiling measurements for HLA-A and the NK ligand PVRL2 compared with temporal protein expression measured [ 12 ]. B. Expression of FAT1 and FAT4 measured by ribosome profiling, RNA-seq and real-time PCR analysis compared with protein abundance [ 12 ]. C and E. Ribosome profiling measurements of BTN2A1 and IGSF8 compared with protein abundance [ 12 ], respectively. D and F. Real-time PCR analysis of btn2a1 (D) and igsf8 (F) mRNA levels along HCMV infection (upper panels). HFF cells stably expressing BTN2A1-HA or IGSF8-HA were infected with HCMV. Protein levels were detected by western blot analysis using anti-HA antibody (lower panels). GFP levels (expressed from the same vector) were used as internal control. Real-time PCR data was normalized by the amount of mfge8 mRNA. Each experiment was performed in triplicates.
    Figure Legend Snippet: Integration between ribosome footprints and protein abundance allows detection of immune ligands that are degraded during infection. A. Ribosome profiling measurements for HLA-A and the NK ligand PVRL2 compared with temporal protein expression measured [ 12 ]. B. Expression of FAT1 and FAT4 measured by ribosome profiling, RNA-seq and real-time PCR analysis compared with protein abundance [ 12 ]. C and E. Ribosome profiling measurements of BTN2A1 and IGSF8 compared with protein abundance [ 12 ], respectively. D and F. Real-time PCR analysis of btn2a1 (D) and igsf8 (F) mRNA levels along HCMV infection (upper panels). HFF cells stably expressing BTN2A1-HA or IGSF8-HA were infected with HCMV. Protein levels were detected by western blot analysis using anti-HA antibody (lower panels). GFP levels (expressed from the same vector) were used as internal control. Real-time PCR data was normalized by the amount of mfge8 mRNA. Each experiment was performed in triplicates.

    Techniques Used: Infection, Expressing, RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Stable Transfection, Western Blot, Plasmid Preparation

    17) Product Images from "New Insights in the Contribution of Voltage-Gated Nav Channels to Rat Aorta Contraction"

    Article Title: New Insights in the Contribution of Voltage-Gated Nav Channels to Rat Aorta Contraction

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0007360

    Identification of Na v channels in the rat aorta and isolated vascular myocytes. (A) Western blot analysis of protein extracts from control tissues (brain; 10 µg of total protein extract per tissue) and aorta (50 µg of total protein extract per tissue) performed using an anti-Pan Na v channel antibody. Labeling specificity was assessed in the brain by pre-incubating the primary antibody with the peptide antigen. Cellular protein content was estimated from the GAPDH signal (lower panel). Arrows indicate the migration of the molecular weight marker (220 and 36 kDa). (B) Analysis of Na v transcripts in total RNA extracted from rat aorta, aortic media and freshly isolated SMCs: representative images obtained after end-point RT-PCR. β-actin or GAPDH primers were used as positive controls to validate reverse transcription. The panel at right shows a sample quantification of Na v isoform transcripts. The expression of Na v 1.2, 1.3 and 1.5 was evaluated by quantitative real-time RT-PCR. Transcript levels were normalized to that of the GAPDH housekeeping gene in each sample and compared in the whole aorta, media layer and freshly isolated SMCs. Data are expressed as means ±s.e.m. of five experiments, each performed in triplicate. *p
    Figure Legend Snippet: Identification of Na v channels in the rat aorta and isolated vascular myocytes. (A) Western blot analysis of protein extracts from control tissues (brain; 10 µg of total protein extract per tissue) and aorta (50 µg of total protein extract per tissue) performed using an anti-Pan Na v channel antibody. Labeling specificity was assessed in the brain by pre-incubating the primary antibody with the peptide antigen. Cellular protein content was estimated from the GAPDH signal (lower panel). Arrows indicate the migration of the molecular weight marker (220 and 36 kDa). (B) Analysis of Na v transcripts in total RNA extracted from rat aorta, aortic media and freshly isolated SMCs: representative images obtained after end-point RT-PCR. β-actin or GAPDH primers were used as positive controls to validate reverse transcription. The panel at right shows a sample quantification of Na v isoform transcripts. The expression of Na v 1.2, 1.3 and 1.5 was evaluated by quantitative real-time RT-PCR. Transcript levels were normalized to that of the GAPDH housekeeping gene in each sample and compared in the whole aorta, media layer and freshly isolated SMCs. Data are expressed as means ±s.e.m. of five experiments, each performed in triplicate. *p

    Techniques Used: Isolation, Western Blot, Antibody Labeling, Migration, Molecular Weight, Marker, Reverse Transcription Polymerase Chain Reaction, Expressing, Quantitative RT-PCR

    18) Product Images from "Activation of Oncogenic Super-Enhancers Is Coupled with DNA Repair by RAD51"

    Article Title: Activation of Oncogenic Super-Enhancers Is Coupled with DNA Repair by RAD51

    Journal: Cell Reports

    doi: 10.1016/j.celrep.2019.09.001

    TEAD4 and RAD51 at Super-enhancers Co-regulate Induction and Repair of DSBs (A) RAD51 binding sites at super, clustered, and single enhancers grouped into two clusters: RAD51/TEAD4 common sites and RAD51 unique binding sites. The graphs in the upper panel show the accumulation of RAD51 and TEAD4 in the common cluster (blue trace) and in the unique cluster (light green trace). (B and C) The distribution of DSBs from siCtrl, siRAD51, and siXRCC4 BLISS samples (B) and from siCtrl and siTEAD4 BLISS samples (C) across the RAD51/TEAD4 common sites cluster (blue trace) and RAD51 unique cluster (light green trace) for each of the three types of enhancers. The red arrows mark the increase of DSBs in siRAD51 and siTEAD4 specifically at RAD51/TEAD4 common sites within super-enhancers. To verify these results, we repeated depletion of RAD51 and TEAD4 twice, and the analysis showed similar increase in DSBs in RAD51/TEAD4 common sites within super-enhancers. (D and E) qRT-PCR demonstrates downregulation of super-enhancer-regulated oncogenes upon depletion of RAD51 (D) and TEAD4 (E) relative to siCtrl samples. (F and G) Inhibition of RAD51 or TEAD4 by B02 or super-TDU, respectively, shows similar decrease of gene expression (F) as well as their eRNAs (G). The qRT-PCR values in (D)–(G) represent mean ± SD from three independent biological samples with technical triplicates. (H) Total RNA-seq around RAD51/TEAD4 common (blue) and RAD51 unique (green) sites within super-enhancers demonstrates downregulation of eRNAs around the common sites upon B02 treatment.
    Figure Legend Snippet: TEAD4 and RAD51 at Super-enhancers Co-regulate Induction and Repair of DSBs (A) RAD51 binding sites at super, clustered, and single enhancers grouped into two clusters: RAD51/TEAD4 common sites and RAD51 unique binding sites. The graphs in the upper panel show the accumulation of RAD51 and TEAD4 in the common cluster (blue trace) and in the unique cluster (light green trace). (B and C) The distribution of DSBs from siCtrl, siRAD51, and siXRCC4 BLISS samples (B) and from siCtrl and siTEAD4 BLISS samples (C) across the RAD51/TEAD4 common sites cluster (blue trace) and RAD51 unique cluster (light green trace) for each of the three types of enhancers. The red arrows mark the increase of DSBs in siRAD51 and siTEAD4 specifically at RAD51/TEAD4 common sites within super-enhancers. To verify these results, we repeated depletion of RAD51 and TEAD4 twice, and the analysis showed similar increase in DSBs in RAD51/TEAD4 common sites within super-enhancers. (D and E) qRT-PCR demonstrates downregulation of super-enhancer-regulated oncogenes upon depletion of RAD51 (D) and TEAD4 (E) relative to siCtrl samples. (F and G) Inhibition of RAD51 or TEAD4 by B02 or super-TDU, respectively, shows similar decrease of gene expression (F) as well as their eRNAs (G). The qRT-PCR values in (D)–(G) represent mean ± SD from three independent biological samples with technical triplicates. (H) Total RNA-seq around RAD51/TEAD4 common (blue) and RAD51 unique (green) sites within super-enhancers demonstrates downregulation of eRNAs around the common sites upon B02 treatment.

    Techniques Used: Binding Assay, Quantitative RT-PCR, Inhibition, Expressing, RNA Sequencing Assay

    19) Product Images from "Changes in the Expression of miR-381 and miR-495 Are Inversely Associated with the Expression of the MDR1 Gene and Development of Multi-Drug Resistance"

    Article Title: Changes in the Expression of miR-381 and miR-495 Are Inversely Associated with the Expression of the MDR1 Gene and Development of Multi-Drug Resistance

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0082062

    Expression of primary transcripts in the miR cluster on chromosome 14q32.31. (A) Schematic representation of known EST sites and selected miRs shown in UCSC genome browser (Human Feb. 2009 (GRCh37/hg19)) at chromosome region 14q32.31. EST primer sets A to E are indicated in triangles below each EST. Transcription start sites sourced from FANTOM CAGE tags are shown as arrows on the top of the 14q32.31 region. (B) Expression of selected miRs (miR-494, miR-495, miR-376c, miR-381 and miR-655) in K562 and K562/ADM cells was validated by stem-loop real-time PCR. The Ct values of these miRs were normalized to that of miR-425 whose level of expression was similar in the two cell lines. The relative expression of miR-494, miR-495, miR-376c, miR-381, miR-655 and miR-16 in K562/ADM cells was calculated. The results are shown as the mean ± SD of three independent experiments. (C) PCR products were resolved on a 2% agarose gel. (D) Expression of the primary transcripts in both K562 and K562/ADM was determined by RT-PCR using primers designed against ESTs A to E. The data were normalized to GAPDH. The results are shown as the mean ± SD of three independent experiments. Quantification of the change of expression level in K562/ADM cells relative to K562 cells is shown on the bottom. (E) Expression of three human RNA nuclease genes, Drosha, DGCR8 and Dicer, in both K562 and K562/ADM cells, was determined by real-time PCR. The expression values were normalized to GAPDH, and are shown as the mean ± SD of three independent experiments.
    Figure Legend Snippet: Expression of primary transcripts in the miR cluster on chromosome 14q32.31. (A) Schematic representation of known EST sites and selected miRs shown in UCSC genome browser (Human Feb. 2009 (GRCh37/hg19)) at chromosome region 14q32.31. EST primer sets A to E are indicated in triangles below each EST. Transcription start sites sourced from FANTOM CAGE tags are shown as arrows on the top of the 14q32.31 region. (B) Expression of selected miRs (miR-494, miR-495, miR-376c, miR-381 and miR-655) in K562 and K562/ADM cells was validated by stem-loop real-time PCR. The Ct values of these miRs were normalized to that of miR-425 whose level of expression was similar in the two cell lines. The relative expression of miR-494, miR-495, miR-376c, miR-381, miR-655 and miR-16 in K562/ADM cells was calculated. The results are shown as the mean ± SD of three independent experiments. (C) PCR products were resolved on a 2% agarose gel. (D) Expression of the primary transcripts in both K562 and K562/ADM was determined by RT-PCR using primers designed against ESTs A to E. The data were normalized to GAPDH. The results are shown as the mean ± SD of three independent experiments. Quantification of the change of expression level in K562/ADM cells relative to K562 cells is shown on the bottom. (E) Expression of three human RNA nuclease genes, Drosha, DGCR8 and Dicer, in both K562 and K562/ADM cells, was determined by real-time PCR. The expression values were normalized to GAPDH, and are shown as the mean ± SD of three independent experiments.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction

    20) Product Images from "Proteomics analysis of bladder cancer invasion: Targeting EIF3D for therapeutic intervention"

    Article Title: Proteomics analysis of bladder cancer invasion: Targeting EIF3D for therapeutic intervention

    Journal: Oncotarget

    doi: 10.18632/oncotarget.17279

    Evaluation of EIF3D knockdown in T24M cells at the RNA and protein level ( A ) Bar graph representing the downregulation of EIF3D in T24M shEIF3D cells in comparison to T24M shscramble and untransduced T24M cells analysed by real-time PCR. The data were normalized to the human GAPDH reference gene and then to the control T24M untransduced cells. ( B ) Western blot analysis for EIF3D in cell extracts derived from T24M, T24M shscramble and T24M shEIF3D cells. ( C ) Bar chart showing data from the quantification analysis of EIF3D protein bands detected in T24M, T24M shscramble and T24M shEIF3D. The quantification of the proteins was performed by using the Quantity One software (BioRad) and the results were normalized to β-Actin loading control and then to the T24M untransduced cells. The values represent the means ± SD from three independent experiments performed in duplicate (two-tailed Student’s t -test, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001).
    Figure Legend Snippet: Evaluation of EIF3D knockdown in T24M cells at the RNA and protein level ( A ) Bar graph representing the downregulation of EIF3D in T24M shEIF3D cells in comparison to T24M shscramble and untransduced T24M cells analysed by real-time PCR. The data were normalized to the human GAPDH reference gene and then to the control T24M untransduced cells. ( B ) Western blot analysis for EIF3D in cell extracts derived from T24M, T24M shscramble and T24M shEIF3D cells. ( C ) Bar chart showing data from the quantification analysis of EIF3D protein bands detected in T24M, T24M shscramble and T24M shEIF3D. The quantification of the proteins was performed by using the Quantity One software (BioRad) and the results were normalized to β-Actin loading control and then to the T24M untransduced cells. The values represent the means ± SD from three independent experiments performed in duplicate (two-tailed Student’s t -test, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001).

    Techniques Used: Real-time Polymerase Chain Reaction, Western Blot, Derivative Assay, Software, Two Tailed Test

    21) Product Images from "HUWE1 controls the development of non-small cell lung cancer through down-regulation of p53"

    Article Title: HUWE1 controls the development of non-small cell lung cancer through down-regulation of p53

    Journal: Theranostics

    doi: 10.7150/thno.24401

    p53 is the key mediator of HUWE1 in lung cancer. (A) The main substrates of HUWE1 were detected by western blotting in HUWE1-wild type and HUWE1-null cells; β-actin was used as a loading control. (B) Total RNA was isolated from the indicated cells. Real-time quantitative RT-PCR was used to determine the expression of genes transcriptionally regulated by p53 in HUWE1-wild type cells and HUWE1-null cells. The genes with fold change > 2 are marked by asterisks. The data are presented as the means ± SD. (C) Immunoblotting was used to determine the expression of cell-cycle associated proteins in the lysates of HUWE1-wild type and HUWE1-null cells; β-actin was used as a loading control. (D) Proteins were extracted from the tumors formed by the HUWE1-intact clone A549 AA1 and the HUWE1-null clone A549 AD11 in BALB/c nude mice, as described in Figure 3 A. Markers of angiogenesis in the lysates were detected by western blotting; #21, #22, #29, and #30 indicate the serial numbers of mice. β-actin was used as a loading control.
    Figure Legend Snippet: p53 is the key mediator of HUWE1 in lung cancer. (A) The main substrates of HUWE1 were detected by western blotting in HUWE1-wild type and HUWE1-null cells; β-actin was used as a loading control. (B) Total RNA was isolated from the indicated cells. Real-time quantitative RT-PCR was used to determine the expression of genes transcriptionally regulated by p53 in HUWE1-wild type cells and HUWE1-null cells. The genes with fold change > 2 are marked by asterisks. The data are presented as the means ± SD. (C) Immunoblotting was used to determine the expression of cell-cycle associated proteins in the lysates of HUWE1-wild type and HUWE1-null cells; β-actin was used as a loading control. (D) Proteins were extracted from the tumors formed by the HUWE1-intact clone A549 AA1 and the HUWE1-null clone A549 AD11 in BALB/c nude mice, as described in Figure 3 A. Markers of angiogenesis in the lysates were detected by western blotting; #21, #22, #29, and #30 indicate the serial numbers of mice. β-actin was used as a loading control.

    Techniques Used: Western Blot, Isolation, Quantitative RT-PCR, Expressing, Mouse Assay

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    Article Snippet: .. RNA extraction and quantitative real-time PCR Total RNA was isolated from individual Arabidopsis organs and seedlings using TRI Reagent (Sigma). ..

    Article Title: Synergism between cAMP and PPAR γ Signalling in the Initiation of UCP1 Gene Expression in HIB1B Brown Adipocytes
    Article Snippet: .. Real-Time PCR Total RNA was extracted from cultured cells using TRI reagent (Sigma). .. Prior to RT-PCR, samples were treated with RNase-free DNase to remove contaminating genomic or plasmid DNA. cDNA was generated using the cDNA synthesis kit from Qiagen.

    Article Title: Interplay between PTB and miR-1285 at the p53 3′UTR modulates the levels of p53 and its isoform Δ40p53α
    Article Snippet: .. RNA isolation and real-time PCR Total RNA was extracted using TRIzol reagent (Sigma). ..

    Isolation:

    Article Title: miR-542-5p Attenuates Fibroblast Activation by Targeting Integrin α6 in Silica-Induced Pulmonary Fibrosis
    Article Snippet: .. RNA Isolation and Quantitative Real-Time PCR Total RNA was extracted using TRIzol reagent (Sigma-Aldrich, T9424, St. Louis, MO, USA), according to the manufacturer’s instructions. .. The concentration and quality of the RNA were confirmed using a Thermo NanoDrop 2000 spectrophotometer (Waltham, MA, USA).

    Article Title: Modulation of the colon cancer cell phenotype by pro-inflammatory macrophages: A preclinical model of surgery-associated inflammation and tumor recurrence
    Article Snippet: .. RNA isolation and Real Time PCR Total RNA was isolated using the GenElute Mammalian Total RNA Kit (Sigma, USA) following the manufacturer instructions, including further DNAse I digestion with the DNA-free Kit (Ambion, USA) to remove traces of genomic DNA. cDNA was obtained using the TaqMan Reverse Transcription Reagents kit (Aplied Biosystems, USA). .. Relative quantification of cDNA was performed by real time polymerase chain reaction (RT-PCR) using the Power SYBR Green PCR Master Mix (Applied Biosystems, USA).

    Article Title: Aberrant KDM5B expression promotes aggressive breast cancer through MALAT1 overexpression and downregulation of hsa-miR-448
    Article Snippet: .. RNA extraction, RT-PCR and real time PCR Total RNA was isolated using TRI Reagent (Sigma) according to manufacturer’s protocol. ..

    Article Title: Arabidopsis SKP1-like protein13 (ASK13) positively regulates seed germination and seedling growth under abiotic stress
    Article Snippet: .. RNA extraction and quantitative real-time PCR Total RNA was isolated from individual Arabidopsis organs and seedlings using TRI Reagent (Sigma). ..

    Article Title: Interplay between PTB and miR-1285 at the p53 3′UTR modulates the levels of p53 and its isoform Δ40p53α
    Article Snippet: .. RNA isolation and real-time PCR Total RNA was extracted using TRIzol reagent (Sigma). ..

    RNA Extraction:

    Article Title: Aberrant KDM5B expression promotes aggressive breast cancer through MALAT1 overexpression and downregulation of hsa-miR-448
    Article Snippet: .. RNA extraction, RT-PCR and real time PCR Total RNA was isolated using TRI Reagent (Sigma) according to manufacturer’s protocol. ..

    Article Title: Arabidopsis SKP1-like protein13 (ASK13) positively regulates seed germination and seedling growth under abiotic stress
    Article Snippet: .. RNA extraction and quantitative real-time PCR Total RNA was isolated from individual Arabidopsis organs and seedlings using TRI Reagent (Sigma). ..

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Aberrant KDM5B expression promotes aggressive breast cancer through MALAT1 overexpression and downregulation of hsa-miR-448
    Article Snippet: .. RNA extraction, RT-PCR and real time PCR Total RNA was isolated using TRI Reagent (Sigma) according to manufacturer’s protocol. ..

    Cell Culture:

    Article Title: Synergism between cAMP and PPAR γ Signalling in the Initiation of UCP1 Gene Expression in HIB1B Brown Adipocytes
    Article Snippet: .. Real-Time PCR Total RNA was extracted from cultured cells using TRI reagent (Sigma). .. Prior to RT-PCR, samples were treated with RNase-free DNase to remove contaminating genomic or plasmid DNA. cDNA was generated using the cDNA synthesis kit from Qiagen.

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  • 94
    Millipore monoclonal anti p53
    Integration of chromatin immunoprecipitation on DNA chip and gene expression profiling . (a) Small interfering RNA (siRNA)-mediated knockdown of <t>p53</t> and/or p73 in HCT16-3(6) cells. Immunoblot of lysates from untreated or hydroxyurea-treated HCT116-3(6) cells (1 mM, 16 hours) following transfection with chemically synthesized siRNA oligos against lacZ, p53, and/or p73. (b) Flow chart for the integrated analysis of chromatin immunoprecipitation on DNA chip with expression profiling. (c) Summary of the relationship between p53/p73 binding and gene expression. Column 4: expression in p53 and p73 siRNA-transfected cells is lower than that of lacZ-transfected cells. Column 5: expression in p53 and p73 siRNA-transfected cells is higher than that of lacZ-transfected cells.
    Monoclonal Anti P53, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monoclonal anti p53/product/Millipore
    Average 94 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    monoclonal anti p53 - by Bioz Stars, 2020-08
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    93
    Millipore rt pcr analysis total rna
    Identification of direct notch targets using a GSI washout experiment . Jurkat cells were treated with 10 uM GSI (or DMSO (untreated)) for 48 hours to accumulate cell surface Notch before washing to permit Notch signalling. After washing, cells were treated with 20 uM cyclohexamide (CHX) or ethanol (vehicle control) to inhibit protein synthesis. After 4 hrs, <t>RNA</t> was isolated and cDNA made for real-time <t>PCR</t> analysis of known Notch target genes (A) and novel Notch target genes (B). Expression values were calculated using cDNA from untreated cells as the calibrator sample. * represents p
    Rt Pcr Analysis Total Rna, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 54 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rt pcr analysis total rna/product/Millipore
    Average 93 stars, based on 54 article reviews
    Price from $9.99 to $1999.99
    rt pcr analysis total rna - by Bioz Stars, 2020-08
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    94
    Millipore mrna expression semi quantitative pcr total rna
    Semi-quantitative <t>PCR</t> analysis of <t>mRNA</t> expression levels of interleukin-6 (IL-6) ( a ), interleukin-1β (IL1–β) ( b ), tumor necrosis factor-α (TNF-α) ( c ), nuclear factor erythroid 2-related factor-2 (Nrf2) ( d ), urocortin-1 (Ucn1) ( e ), glutathione peroxidase-1 (Gpx-1) ( f ) and β-actin ( g ) in the liver of control and hyperandrogenic PCOM rats.
    Mrna Expression Semi Quantitative Pcr Total Rna, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mrna expression semi quantitative pcr total rna/product/Millipore
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mrna expression semi quantitative pcr total rna - by Bioz Stars, 2020-08
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    Integration of chromatin immunoprecipitation on DNA chip and gene expression profiling . (a) Small interfering RNA (siRNA)-mediated knockdown of p53 and/or p73 in HCT16-3(6) cells. Immunoblot of lysates from untreated or hydroxyurea-treated HCT116-3(6) cells (1 mM, 16 hours) following transfection with chemically synthesized siRNA oligos against lacZ, p53, and/or p73. (b) Flow chart for the integrated analysis of chromatin immunoprecipitation on DNA chip with expression profiling. (c) Summary of the relationship between p53/p73 binding and gene expression. Column 4: expression in p53 and p73 siRNA-transfected cells is lower than that of lacZ-transfected cells. Column 5: expression in p53 and p73 siRNA-transfected cells is higher than that of lacZ-transfected cells.

    Journal: BMC Biology

    Article Title: Effect of hydroxyurea on the promoter occupancy profiles of tumor suppressor p53 and p73

    doi: 10.1186/1741-7007-7-35

    Figure Lengend Snippet: Integration of chromatin immunoprecipitation on DNA chip and gene expression profiling . (a) Small interfering RNA (siRNA)-mediated knockdown of p53 and/or p73 in HCT16-3(6) cells. Immunoblot of lysates from untreated or hydroxyurea-treated HCT116-3(6) cells (1 mM, 16 hours) following transfection with chemically synthesized siRNA oligos against lacZ, p53, and/or p73. (b) Flow chart for the integrated analysis of chromatin immunoprecipitation on DNA chip with expression profiling. (c) Summary of the relationship between p53/p73 binding and gene expression. Column 4: expression in p53 and p73 siRNA-transfected cells is lower than that of lacZ-transfected cells. Column 5: expression in p53 and p73 siRNA-transfected cells is higher than that of lacZ-transfected cells.

    Article Snippet: Approximately 3 × 107 cells were prepared per immunoprecipitation and incubated with 5 μg of total monoclonal anti-p53 (1:1 mixtures of Ab-1 and Ab-12, CalBiochem) or affinity-purified polyclonal anit-p73 (827) at 4°C overnight.

    Techniques: Chromatin Immunoprecipitation, Expressing, Small Interfering RNA, Transfection, Synthesized, Flow Cytometry, Binding Assay

    Model-based analysis for promoter array . (a) Scheme for chromatin immunoprecipitation (ChIP) on DNA chip analysis. (b) Confirmation of ChIP and ligation-mediated -polymerase chain reaction amplified amplicons. ChIPs were performed as described in Figure 1(e). Enrichment of the p21cip1 distal promoter region was used to check the specificity of the samples prior to hybridization, M = mouse immunoglobulin (IgG); R = rabbit IgG. (c) Outline of the bioinformatics analysis for the identification of p53 and p73 ChIP-enriched promoters. (d) Definition of window and promoter-scores. a-g: window score; *: promoter-score. (e) Pair-wise scatter plots of the promoter-scores for the input channels (top). R 2 values are summarized in the table. (f) Pair-wise scatter plots of the promoter-scores between the ChIP and input channels (top). R 2 values are summarized in the table. (g) Pair-wise scatter plots of the promoter-scores between the ChIP and IgG channels (top). R 2 values are summarized in the table. (h) Correlation between the ChIP channels and OCT4 ChIP control. R 2 values are summarized in the table.

    Journal: BMC Biology

    Article Title: Effect of hydroxyurea on the promoter occupancy profiles of tumor suppressor p53 and p73

    doi: 10.1186/1741-7007-7-35

    Figure Lengend Snippet: Model-based analysis for promoter array . (a) Scheme for chromatin immunoprecipitation (ChIP) on DNA chip analysis. (b) Confirmation of ChIP and ligation-mediated -polymerase chain reaction amplified amplicons. ChIPs were performed as described in Figure 1(e). Enrichment of the p21cip1 distal promoter region was used to check the specificity of the samples prior to hybridization, M = mouse immunoglobulin (IgG); R = rabbit IgG. (c) Outline of the bioinformatics analysis for the identification of p53 and p73 ChIP-enriched promoters. (d) Definition of window and promoter-scores. a-g: window score; *: promoter-score. (e) Pair-wise scatter plots of the promoter-scores for the input channels (top). R 2 values are summarized in the table. (f) Pair-wise scatter plots of the promoter-scores between the ChIP and input channels (top). R 2 values are summarized in the table. (g) Pair-wise scatter plots of the promoter-scores between the ChIP and IgG channels (top). R 2 values are summarized in the table. (h) Correlation between the ChIP channels and OCT4 ChIP control. R 2 values are summarized in the table.

    Article Snippet: Approximately 3 × 107 cells were prepared per immunoprecipitation and incubated with 5 μg of total monoclonal anti-p53 (1:1 mixtures of Ab-1 and Ab-12, CalBiochem) or affinity-purified polyclonal anit-p73 (827) at 4°C overnight.

    Techniques: Chromatin Immunoprecipitation, Ligation, Polymerase Chain Reaction, Amplification, Hybridization

    Comparison of p53 and p73 promoter occupancy profiles . (a) Summary of the correlation analysis on selected promoters using model-based algorithm for promoter arrays promoter-scores (column 3), or NimbleGen log2 ratios (column 4). R 2 values represent the degree of correlation and P values represent the significance of correlation. (b) Venn diagrams depicting the number of common and distinct promoters occupied by p53 and/or p73 using FDR MAP

    Journal: BMC Biology

    Article Title: Effect of hydroxyurea on the promoter occupancy profiles of tumor suppressor p53 and p73

    doi: 10.1186/1741-7007-7-35

    Figure Lengend Snippet: Comparison of p53 and p73 promoter occupancy profiles . (a) Summary of the correlation analysis on selected promoters using model-based algorithm for promoter arrays promoter-scores (column 3), or NimbleGen log2 ratios (column 4). R 2 values represent the degree of correlation and P values represent the significance of correlation. (b) Venn diagrams depicting the number of common and distinct promoters occupied by p53 and/or p73 using FDR MAP

    Article Snippet: Approximately 3 × 107 cells were prepared per immunoprecipitation and incubated with 5 μg of total monoclonal anti-p53 (1:1 mixtures of Ab-1 and Ab-12, CalBiochem) or affinity-purified polyclonal anit-p73 (827) at 4°C overnight.

    Techniques:

    Summary of quantitative chromatin immunoprecipitation analysis of selected promoters for p53 and p73 binding before and after hydroxyurea treatment . Cross-linked chromatin from untreated hydroxyurea (-HU) or hydroxyurea-treated (+HU) (1 mM, 16 hours) HCT116-3(6) cells were immunoprecipitated with the indicated antibodies and followed by polymerase chain reaction analysis using the primers flanking the sequences corresponding to the model-based algorithm for promoter array promoter-score window in each of the promoters. Data shown are log2 occupancy units from two independent chromatin immunoprecipitation experiments +/- standard deviation. log2 occupancy units > 1: significant enrichment of the selected promoter sequences in the anti-p53 or anti-p73 chromatin immunoprecipitation.

    Journal: BMC Biology

    Article Title: Effect of hydroxyurea on the promoter occupancy profiles of tumor suppressor p53 and p73

    doi: 10.1186/1741-7007-7-35

    Figure Lengend Snippet: Summary of quantitative chromatin immunoprecipitation analysis of selected promoters for p53 and p73 binding before and after hydroxyurea treatment . Cross-linked chromatin from untreated hydroxyurea (-HU) or hydroxyurea-treated (+HU) (1 mM, 16 hours) HCT116-3(6) cells were immunoprecipitated with the indicated antibodies and followed by polymerase chain reaction analysis using the primers flanking the sequences corresponding to the model-based algorithm for promoter array promoter-score window in each of the promoters. Data shown are log2 occupancy units from two independent chromatin immunoprecipitation experiments +/- standard deviation. log2 occupancy units > 1: significant enrichment of the selected promoter sequences in the anti-p53 or anti-p73 chromatin immunoprecipitation.

    Article Snippet: Approximately 3 × 107 cells were prepared per immunoprecipitation and incubated with 5 μg of total monoclonal anti-p53 (1:1 mixtures of Ab-1 and Ab-12, CalBiochem) or affinity-purified polyclonal anit-p73 (827) at 4°C overnight.

    Techniques: Chromatin Immunoprecipitation, Binding Assay, Immunoprecipitation, Polymerase Chain Reaction, Standard Deviation

    MLH3 is a p73-specific target induced by hydroxyurea . (a) Western blotting analysis of lysates from untreated or hydroxyurea (HU)-treated HCT116-3(6) cells (1 mM, 16 hours) stably expressing lacZ, p53, or p73 small hairpin RNA (shRNA). (b) Effect of p53 or p73 knockdown on p21cip1 gene expression. Real-time RT-PCR analysis of the steady-state p21cip1 mRNA level in HCT116-3(6) cells stably expressing p53 or p73 shRNA *P

    Journal: BMC Biology

    Article Title: Effect of hydroxyurea on the promoter occupancy profiles of tumor suppressor p53 and p73

    doi: 10.1186/1741-7007-7-35

    Figure Lengend Snippet: MLH3 is a p73-specific target induced by hydroxyurea . (a) Western blotting analysis of lysates from untreated or hydroxyurea (HU)-treated HCT116-3(6) cells (1 mM, 16 hours) stably expressing lacZ, p53, or p73 small hairpin RNA (shRNA). (b) Effect of p53 or p73 knockdown on p21cip1 gene expression. Real-time RT-PCR analysis of the steady-state p21cip1 mRNA level in HCT116-3(6) cells stably expressing p53 or p73 shRNA *P

    Article Snippet: Approximately 3 × 107 cells were prepared per immunoprecipitation and incubated with 5 μg of total monoclonal anti-p53 (1:1 mixtures of Ab-1 and Ab-12, CalBiochem) or affinity-purified polyclonal anit-p73 (827) at 4°C overnight.

    Techniques: Western Blot, Stable Transfection, Expressing, shRNA, Quantitative RT-PCR

    Hydroxyurea-induced accumulation of p53 and p73 and increased occupancy of the p21cip1 promoter in HCT116-3(6) cells . (a) The indicated cells were treated with or without HU (1 mM) for 16 hours, and the indicated proteins were detected by immunoblotting as described in Methods. (b) Higher levels of p53 and p73 induced by HU correlates with increased association with chromatin. HCT116-3(6) cells with or without HU treatment were subjected to chromatin immunoprecipitation (ChIP) using the indicated antibodies followed by immunoblotting with the indicated antibodies. (c) Polymerase chain reaction (PCR) primer designed for the distal p53 binding site in the p21cip1 promoter. (d) ChIP specificity. p73 -/- 3T3 cells reconstituted with either the empty vector or human p73α were treated with or without HU as in (a) and were subjected to ChIP using the indicated antibodies. Enrichment of the p21cip1 promoters was assessed by PCR using primers encompassing the p53-binding site in the distal region of the p21cip1 promoter as shown in (c). (e) Increased occupancy of the p21cip1 promoter by p53 and p73 following HU treatment. Chromatin from HCT116-3(6) cells with or without HU treatment were immunoprecipitated with the indicated antibodies (lanes 2, 4, 6, and 8) and followed by PCR analysis using the primers as shown in (c). M = mouse immunoglobulin G (IgG); R = rabbit IgG. Fold enrichment relative to the IgG sample was determined by quantitative PCR (f) Quantitative real time-PCR analysis of (e) shown as percent total input DNA.

    Journal: BMC Biology

    Article Title: Effect of hydroxyurea on the promoter occupancy profiles of tumor suppressor p53 and p73

    doi: 10.1186/1741-7007-7-35

    Figure Lengend Snippet: Hydroxyurea-induced accumulation of p53 and p73 and increased occupancy of the p21cip1 promoter in HCT116-3(6) cells . (a) The indicated cells were treated with or without HU (1 mM) for 16 hours, and the indicated proteins were detected by immunoblotting as described in Methods. (b) Higher levels of p53 and p73 induced by HU correlates with increased association with chromatin. HCT116-3(6) cells with or without HU treatment were subjected to chromatin immunoprecipitation (ChIP) using the indicated antibodies followed by immunoblotting with the indicated antibodies. (c) Polymerase chain reaction (PCR) primer designed for the distal p53 binding site in the p21cip1 promoter. (d) ChIP specificity. p73 -/- 3T3 cells reconstituted with either the empty vector or human p73α were treated with or without HU as in (a) and were subjected to ChIP using the indicated antibodies. Enrichment of the p21cip1 promoters was assessed by PCR using primers encompassing the p53-binding site in the distal region of the p21cip1 promoter as shown in (c). (e) Increased occupancy of the p21cip1 promoter by p53 and p73 following HU treatment. Chromatin from HCT116-3(6) cells with or without HU treatment were immunoprecipitated with the indicated antibodies (lanes 2, 4, 6, and 8) and followed by PCR analysis using the primers as shown in (c). M = mouse immunoglobulin G (IgG); R = rabbit IgG. Fold enrichment relative to the IgG sample was determined by quantitative PCR (f) Quantitative real time-PCR analysis of (e) shown as percent total input DNA.

    Article Snippet: Approximately 3 × 107 cells were prepared per immunoprecipitation and incubated with 5 μg of total monoclonal anti-p53 (1:1 mixtures of Ab-1 and Ab-12, CalBiochem) or affinity-purified polyclonal anit-p73 (827) at 4°C overnight.

    Techniques: Chromatin Immunoprecipitation, Polymerase Chain Reaction, Binding Assay, Plasmid Preparation, Immunoprecipitation, Real-time Polymerase Chain Reaction

    Identification of direct notch targets using a GSI washout experiment . Jurkat cells were treated with 10 uM GSI (or DMSO (untreated)) for 48 hours to accumulate cell surface Notch before washing to permit Notch signalling. After washing, cells were treated with 20 uM cyclohexamide (CHX) or ethanol (vehicle control) to inhibit protein synthesis. After 4 hrs, RNA was isolated and cDNA made for real-time PCR analysis of known Notch target genes (A) and novel Notch target genes (B). Expression values were calculated using cDNA from untreated cells as the calibrator sample. * represents p

    Journal: Molecular Cancer

    Article Title: Identification of novel Notch target genes in T cell leukaemia

    doi: 10.1186/1476-4598-8-35

    Figure Lengend Snippet: Identification of direct notch targets using a GSI washout experiment . Jurkat cells were treated with 10 uM GSI (or DMSO (untreated)) for 48 hours to accumulate cell surface Notch before washing to permit Notch signalling. After washing, cells were treated with 20 uM cyclohexamide (CHX) or ethanol (vehicle control) to inhibit protein synthesis. After 4 hrs, RNA was isolated and cDNA made for real-time PCR analysis of known Notch target genes (A) and novel Notch target genes (B). Expression values were calculated using cDNA from untreated cells as the calibrator sample. * represents p

    Article Snippet: RT-PCR analysis Total RNA was isolated from GFP+ transduced cell lines or cells treated with gamma secretase inhibitor (GSI IX; Calbiochem) and reverse transcribed to cDNA using the High Capacity cDNA Archive kit (Applied Biosystems, Warrington, UK).

    Techniques: Isolation, Real-time Polymerase Chain Reaction, Expressing

    Semi-quantitative PCR analysis of mRNA expression levels of interleukin-6 (IL-6) ( a ), interleukin-1β (IL1–β) ( b ), tumor necrosis factor-α (TNF-α) ( c ), nuclear factor erythroid 2-related factor-2 (Nrf2) ( d ), urocortin-1 (Ucn1) ( e ), glutathione peroxidase-1 (Gpx-1) ( f ) and β-actin ( g ) in the liver of control and hyperandrogenic PCOM rats.

    Journal: Medicina

    Article Title: Effect of DHT-Induced Hyperandrogenism on the Pro-Inflammatory Cytokines in a Rat Model of Polycystic Ovary Morphology

    doi: 10.3390/medicina56030100

    Figure Lengend Snippet: Semi-quantitative PCR analysis of mRNA expression levels of interleukin-6 (IL-6) ( a ), interleukin-1β (IL1–β) ( b ), tumor necrosis factor-α (TNF-α) ( c ), nuclear factor erythroid 2-related factor-2 (Nrf2) ( d ), urocortin-1 (Ucn1) ( e ), glutathione peroxidase-1 (Gpx-1) ( f ) and β-actin ( g ) in the liver of control and hyperandrogenic PCOM rats.

    Article Snippet: Analysis of mRNA Expression (Semi-Quantitative PCR) Total RNA was isolated from control and DHT-treated PCOM rat liver using the TRIzol reagent (monophasic solution of phenol and guanidinium isothiocyanate; Cat # 93289, Millipore Sigma, Burlington, MA, USA) at indicated time points.

    Techniques: Real-time Polymerase Chain Reaction, Expressing